Image analysis and measurement of biological samples

ABSTRACT

Methods, devices, systems, and apparatuses are provided for the image analysis of measurement of biological samples.

This application claims priority to, and the benefit under 35 U.S.C.§119(e) of, U.S. Patent Application Ser. No. 61/675,811, filed Jul. 25,2012; U.S. Pat. App. Ser. No. 61/676,178, filed Jul. 26, 2012; U.S.Patent Application 61/766,116, filed Feb. 18, 2013; and U.S. PatentApplication 61/802,194, filed Mar. 15, 2013; the disclosures of all ofwhich patent applications are hereby fully incorporated by reference intheir entireties for all purposes.

BACKGROUND

Analysis of biological samples from a subject may be important forhealth-related diagnosing, monitoring and/or treating of the subject. Avariety of methods are known for the analysis of biological samples.However, in order to provide better diagnosing, monitoring, and/ortreating of subjects, improvements in the analysis of biological samplesare desired.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

COPYRIGHT

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to facsimilereproduction of the patent documents and disclosures, as they appear inthe US Patent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever. The following notice shallapply: Copyright 2013 Theranos, Inc.

SUMMARY

Methods, devices, systems, and apparatuses are described herein forimage analysis and/or measurement of biological samples.

In one embodiment, a method for the measurement of a component ofinterest in cells of a cellular population in a sample is provided,including: a) obtaining a quantitative measurement of a marker presentin cells of the cellular population in the sample; b) based on themeasurement of part a), determining, with the aid of a computer, anapproximate amount of cells in the cellular population present in thesample; c) based on the results of part b), selecting an amount ofreagent to add to the sample, wherein the reagent binds specifically tothe component of interest in cells of the cellular population and isconfigured to be readily detectable; d) based on the results of part c),adding the selected amount of the reagent to the sample; e) assayingcells in the sample for reagent bound to the compound of interest; andf) based on the amount of reagent bound to the compound of interest,determining the amount of the component of interest in cells of thecellular population of the sample. In an embodiment of the method, thereagent of part c) is an antibody.

In another embodiment, a method for focusing a microscope is provided,including: a) mixing a sample containing an object for microscopicanalysis with a reference particle having a known size, to generate amixture containing the sample and reference particle; b) positioning themixture of step a) into a light path of a microscope; c) exposing themixture of step a) to a light beam configured to visualize the referenceparticle; and d) focusing the microscope based on the position of thereference particle within the mixture.

In yet another embodiment, provided herein is a method for identifying acell in a sample containing a plurality of cells, including: a) assayinga cell of the plurality of cells for at least one of: (i) the presenceof a cell surface antigen; (ii) the amount of a cell surface antigen; or(iii) cell size; b) assay the cell of a) for at least one of: (i)nuclear size; or (ii) nuclear shape; and c) assaying the cell of a) andb) for quantitative cell light scatter, wherein the combination ofinformation from steps a), b) and c) is used to identify the cell in thesample containing a plurality of cells.

In yet another embodiment, provided herein is a system of a detectorassembly for use with a sample holder that holds a sample to beexamined. In one non-limiting example, the sample holder is a cuvettethat has features and/or materials in it that enable the cuvette to beengaged and moved from one location to the detector assembly. In someembodiments, the detector assembly has a first surface that isconfigured to engage a surface of the sample holder in a manner suchthat the interface between the two does not create optical interferencein the optical pathway from the detector assembly to the sample in thesample holder. In one embodiment, there may be more than one location onthe detector assembly for one or more of the sample holders. Someembodiments may have the same sample holder for each of the locations.Optionally, some embodiments may have different sample holders for atleast some of the locations associated with the detector assembly.

In one embodiment described herein, a sample holder is provided hereinsuch as but not limited to a cuvette with optical properties,dimensions, materials, and/or physical features that allow for it tohold the sample for analysis by the detector assembly while keeping itphysically separate from and not in direct contact with the detectorassembly. This can be particularly useful for sample fluids that containshaped members therein.

In one embodiment described herein, the detector assembly may be amulti-channel microscopy unit that is configured to obtain shape ofcell, physical, optical, and biochemical properties all in the samedevice. It can provide both quantitative info, and descriptive info. Oneembodiment of the detector assembly may use multiple markers of the samecolor, and then deconvolute signals—this allows reduction in number ofspectral channels and light sources.

It should be understood that some embodiments herein may have a sampleholder such as but not limited to a cuvette with physical features inthe shape of the cuvette material that increase darkfield illuminationwhere some features are for light reflectance, optionally some formechanical support. The system herein can use both epi (direct) andtrans (reflected) illumination in darkfield imaging. This differs fromtraditional darkfield imaging which uses mainly epi, not transillumination. Thus, the combo of epi and trans illumination, wherein thetrans illumination originates from the same light source as the epiillumination differs from known systems. Optionally, the use of a shapedsample holder such as the cuvette can be used to provide the transillumination. Optionally, the trans illumination is at a non-negligablelevel [at least×amount]. Optionally, one embodiment may add an actualreflective surface to increase trans light generated. The dark fieldlight source may be an LED, laser, or other illumination source that canprovide the desired excitation wavelength(s).

In one embodiment, the combination of the microscope objective andringlight (for darkfield microscopy) is at a physical distance betweenthem that enables a compact size for the detector assembly.

In yet another embodiment, information from the cytometry assay, eitherfrom the sample preparation phase and/or from the analysis phase, isused to guide/trigger a secondary procedure. One procedure may be toprovide an alert for direct human review. Another is to use an estimatedcell count or other information from the sample preparation stage to beinformation used to guide assay performance in another procedure.

Techniques for counting cells can also provide ways to deal with unevencuvettes. One method comprises using: a) volume-metered channel tointroduce a known volume into a channel. The method may include countingall cells in the cuvette. Since one knows the volume of sample, one alsoknows the concentration of cells in volume (this may be performed inhydrophobic containers or cuvettes with chambers with such surfaces).Another method comprises: b) ratio-based metric to mix sample with aknown amount of beads, which is used to calculate the concentration ofcells in the sample based on the number of beads observed.

In yet another embodiment described herein, a method is providedcomprising measuring formed blood components such as but not limited toRBC volume by swelling the formed blood components such as but notlimited to RBCs into spheres, and measuring volume by darkfieldmicroscopy.

In yet another embodiment described herein, a method is providedcomprising measuring platelet volume. The method may including labelingplatelets with fluorescent dye and measuring the size of signal. Onethen add beads to sample of known size and compare to platelet.

It should be understood that embodiments in this disclosure may beadapted to have one or more of the features described in thisdisclosure.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

FIG. 1A shows a plot of side scatter intensity (x-axis) vs. fluorescenceintensity of a mixture cells including natural killer cells andneutrophils labeled with a fluorescent binder that recognizes CD16.

FIG. 1B shows a bar graph showing the ratio of nuclear area to totalcell area of natural killer cells (“NK”) and neutrophils (“Neu”).

FIG. 1C shows natural killer cells stained with anti-CD16 antibody (leftcolumn) and a nuclear stain (right column).

FIG. 1D shows neutrophils stained with anti-CD16 antibody (left column)and a nuclear stain (right column).

FIG. 2A shows platelets labeled with fluorescently conjugated CD41 andCD61 antibodies (bright dots).

FIG. 2B shows the intensity distribution of images of fluorescentlylabeled platelets at 10× (left) and 20× (right) magnification.

FIG. 2C shows the intensity distribution of an image of a fluorescentlylabeled platelet showing measured intensity (light grey) and curve fitto the measured intensity (dark grey).

FIG. 3 shows a plot of a curve of showing the relationship between thenominal diameter of standard particles in μm (x-axis) and fluorescenceintensity-based size as measured in arbitrary units (a.u.) (y-axis). Thefigure also shows representative beads at different points along thecurve.

FIG. 4A shows sphered red blood cells imaged by dark field microscopy incuvettes that allow only epi-illumination.

FIG. 4B shows sphered red blood cells imaged by dark field microscopy incuvettes that allow a mixture of epi- and trans-illumination.

FIG. 5A shows putative band neutrophils stained with anti-CD16 antibodyand a nuclear stain.

FIG. 5B shows putative segmented neutrophils stained with anti-CD 16antibody and a nuclear stain.

FIG. 6 shows a schematic perspective view of an embodiment of anexemplary imaging system.

FIG. 7 shows a schematic side view (indicating cross-sections of someelements) of an embodiment of an exemplary imaging system.

FIG. 8A shows a schematic cross-sectional side view an embodiment of anexemplary imaging system.

FIG. 8B shows a schematic perspective view an embodiment of an exemplaryimaging system.

FIG. 8C shows a schematic perspective view an embodiment of an exemplarysystem including an imaging system.

FIG. 9A is a dark-field image which shows representative images of bloodcells taken from whole blood.

FIG. 9B is an image of blood cells taken from whole blood showingfluorescence from labeled anti-CD14 antibodies attached to monocytes.

FIG. 9C is an image of blood cells taken from whole blood showingfluorescence from labeled anti-CD123 antibodies attached to basophils.

FIG. 9D is an image of blood cells taken from whole blood showingfluorescence from labeled anti-CD16 antibodies attached to neutrophils.

FIG. 9E is an image of blood cells taken from whole blood showingfluorescence from labeled anti-CD45 antibodies attached to leukocytes.

FIG. 9F is an image of blood cells taken from whole blood showingleukocyte and platelet cells stained with nuclear stain Draq5 (red bloodcells, lacking nuclei, are not stained by Draq5).

FIG. 10 is composite image which shows representative images of bloodcells taken from whole blood, showing a monocyte, a lymphocyte, aneosinophil, and a neutrophil.

FIG. 11A shows identification of monocytes by plotting intensity ofFL-17 versus FL-9 intensity. Plots of fluorescence detected on cellslabeled with different markers (labeled antibodies directed at differentcell-surface or other markers) are useful for identifying cells.

FIG. 11B shows identification of basophils by plotting intensity ofFL-19 versus FL-15 intensity.

FIG. 11C shows identification of lymphocytes by plotting intensity ofFL-15 versus FL-11 intensity.

FIG. 11D shows identification of neutrophils and eosinophils by plottingintensity of FL-15 versus FL-9 intensity.

FIG. 12A plots white blood cell counts obtained by the present methodsversus white blood cell counts obtained by a commercial blood analyzer.Comparisons of cell counts (measured from aliquots of the same bloodsample) were obtained by the present methods, and were plotted againstcell counts obtained by other methods (using a commercial bloodanalyzer).

FIG. 12B plots red blood cell counts obtained by the present methodsversus red blood cell counts obtained by the commercial blood analyzer.

FIG. 12C plots platelet counts obtained by the present methods versusplatelet counts obtained by the commercial blood analyzer.

FIG. 12D plots neutrophil counts obtained by the present methods versusneutrophil counts obtained by the commercial blood analyzer.

FIG. 12E plots monocyte counts obtained by the present methods versusmonocyte counts obtained by the commercial blood analyzer.

FIG. 12F plots lymphocyte counts obtained by the present methods versuslymphocyte counts obtained by the commercial blood analyzer.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. It may be notedthat, as used in the specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a material”may include mixtures of materials, reference to “a compound” may includemultiple compounds, and the like. References cited herein are herebyincorporated by reference in their entirety, except to the extent thatthey conflict with teachings explicitly set forth in this specification.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, if a device optionally contains a feature for a samplecollection unit, this means that the sample collection unit may or maynot be present, and, thus, the description includes both structureswherein a device possesses the sample collection unit and structureswherein sample collection unit is not present.

As used herein, the terms “substantial” means more than a minimal orinsignificant amount; and “substantially” means more than a minimally orinsignificantly. Thus, for example, the phrase “substantiallydifferent”, as used herein, denotes a sufficiently high degree ofdifference between two numeric values such that one of skill in the artwould consider the difference between the two values to be ofstatistical significance within the context of the characteristicmeasured by said values. Thus, the difference between two values thatare substantially different from each other is typically greater thanabout 10%, and may be greater than about 20%, preferably greater thanabout 30%, preferably greater than about 40%, preferably greater thanabout 50% as a function of the reference value or comparator value.

As used herein, a “sample” may be but is not limited to a blood sample,or a portion of a blood sample, may be of any suitable size or volume,and is preferably of small size or volume. In some embodiments of theassays and methods disclosed herein, measurements may be made using asmall volume blood sample, or no more than a small volume portion of ablood sample, where a small volume comprises no more than about 5 mL; orcomprises no more than about 3 mL; or comprises no more than about 2 mL;or comprises no more than about 1 mL; or comprises no more than about500 μL; or comprises no more than about 250 μL; or comprises no morethan about 100 μL; or comprises no more than about 75 μL; or comprisesno more than about 50 μL; or comprises no more than about 35 μL; orcomprises no more than about 25 μL; or comprises no more than about 20μL; or comprises no more than about 15 μL; or comprises no more thanabout 10 μL; or comprises no more than about 8 μL; or comprises no morethan about 6 μL; or comprises no more than about 5 μL; or comprises nomore than about 4 μL; or comprises no more than about 3 μL; or comprisesno more than about 2 μL; or comprises no more than about 1 μL; orcomprises no more than about 0.8 μL; or comprises no more than about 0.5μL; or comprises no more than about 0.3 μL; or comprises no more thanabout 0.2 μL; or comprises no more than about 0.1 μL; or comprises nomore than about 0.05 μL; or comprises no more than about 0.01 μL.

As used herein, the term “point of service location” may includelocations where a subject may receive a service (e.g. testing,monitoring, treatment, diagnosis, guidance, sample collection, IDverification, medical services, non-medical services, etc.), and mayinclude, without limitation, a subject's home, a subject's business, thelocation of a healthcare provider (e.g., doctor), hospitals, emergencyrooms, operating rooms, clinics, health care professionals' offices,laboratories, retailers [e.g. pharmacies (e.g., retail pharmacy,clinical pharmacy, hospital pharmacy), drugstores, supermarkets,grocers, etc.], transportation vehicles (e.g. car, boat, truck, bus,airplane, motorcycle, ambulance, mobile unit, fire engine/truck,emergency vehicle, law enforcement vehicle, police car, or other vehicleconfigured to transport a subject from one point to another, etc.),traveling medical care units, mobile units, schools, day-care centers,security screening locations, combat locations, health assisted livingresidences, government offices, office buildings, tents, bodily fluidsample acquisition sites (e.g. blood collection centers), sites at ornear an entrance to a location that a subject may wish to access, siteson or near a device that a subject may wish to access (e.g., thelocation of a computer if the subject wishes to access the computer), alocation where a sample processing device receives a sample, or anyother point of service location described elsewhere herein.

The term “cells,” as used in the context of biological samples,encompasses samples that are generally of similar sizes to individualcells, including but not limited to vesicles (such as liposomes), cells,virions, and substances bound to small particles such as beads,nanoparticles, or microspheres.

Quantitative Microscopy

In some embodiments, methods, systems, and devices are provided hereinfor quantitative microscopy. Quantitative microscopy may involve one ormore of quantitative fluorescence microscopy, quantitative dark fieldmicroscopy, quantitative bright field microscopy, and quantitative phasecontrast microscopy methods to measure one or more cellular attributes.Any of these methods may provide morphometric information regardingcells. Such information may be measured quantitatively. In someembodiments, for quantitative microscopy, a sample is analyzed by two ormore of quantitative fluorescence microscopy, quantitative dark fieldmicroscopy, quantitative bright field microscopy, and quantitative phasecontrast microscopy. Quantitative microscopy may include use of imageanalysis techniques and/or statistical learning and classificationmethods to process images obtained by microscopy.

Multiple different cellular attributes may be measured duringquantitative microscopy. Cellular attributes that may be measuredinclude, without limitation:

Physical attributes: e.g. cell size, volume, conductivity, low and highangle scatter, and density.

Morphological attributes: e.g. cell shape, area, size, and lobularity;nucleus shape area, size, and lobularity; mitochondria shape, area,size, and lobularity; and ratio of nuclear volume to cell volume.

Intracellular attributes: e.g. nucleus centroid/cell centroid distance(i.e. distance between the center of the nucleus and the center of thecell), nucleus lobe centroid distance (i.e. distance between the centerof different lobes of the nucleus), distribution of proteins with thecells (e.g. actin, tubulin, etc.), and distribution of organelles withinthe cells (e.g. lysosomes, mitochondria, etc.).

Biochemical attributes: e.g. expression level of cellular proteins, cellsurface proteins, cytoplasmic proteins, nuclear proteins, cellularnucleic acids, cell surface nucleic acids, cytoplasmic nucleic acids,nuclear nucleic acids, cellular carbohydrates, cell surfacecarbohydrates, cytoplasmic carbohydrates, and nuclear carbohydrates.

In some embodiments, methods, systems, and devices are provided hereinfor the quantitative measurement of two, three, four, five or moreattributes of cells in a sample, wherein the attributes are selectedfrom physical attributes, morphological attributes, intracellularattributes, and biochemical attributes. In some embodiments, methods,systems, and devices are provided herein for the quantitativemeasurement of two, three, four, five or more attributes of cells in asample, wherein the attributes are selected from: cell size, cellvolume, cell conductivity, cell low angle light scatter, cell high anglelight scatter, cell density, cell shape, cell area, cell lobularity,nucleus shape, nucleus area, nucleus size, nucleus lobularity,mitochondria shape, mitochondria area, mitochondria size, mitochondrialobularity, ratio of nuclear volume to cell volume, nucleuscentroid/cell centroid distance, nucleus lobe centroid distance,distribution of proteins with the cells (e.g. actin, tubulin, etc.),distribution of organelles within the cells (e.g. lysosomes,mitochondria, etc.), expression level of a cellular protein, expressionlevel of a cell surface protein, expression level of a cytoplasmicprotein, expression level of a nuclear protein, expression level of acellular nucleic acid, expression level of a cell surface nucleic acid,expression level of a cytoplasmic nucleic acid, expression level of anuclear nucleic acid, expression level of a cellular carbohydrate,expression level of a cell surface carbohydrate, expression level of acytoplasmic carbohydrate, and expression level of a nuclearcarbohydrate.

In some embodiments, methods are provided for the quantitativemeasurement of two, three, four, five, or more attributes of cells in abiological sample by microscopy, wherein the method may include one ormore of the following steps or elements. The attributes of the cellsquantitatively measured may be selected from the attributes listed inthe immediately above paragraph. The biological sample may bepre-treated prior to microscopy. Pre-treatment may include any procedureto aid in the analysis of the sample by microscopy, including: treatmentof the sample to enrich for cells of interest for microscopy, treatmentof the sample to reduce components in the sample which may interferewith microscopy, addition of material to the sample to facilitateanalysis of the sample by microscopy (e.g. diluents, blocking moleculesto reduce non-specific binding of dyes to cells, etc.). Optionally,prior to microscopy, a sample may be contacted with one or more bindersthat specifically bind to a cellular component. Binders may be directlylinked to a dye or other particle for the visualization of the binder. Asample may also be contacted with a secondary binder, which binds to thebinder which binds to the cellular component. A secondary binder may bedirectly linked to a dye or other particle for the visualization of thebinder. Prior to microscopy, a sample may be assayed in aspectrophotometer. For microscopy, a biological sample containing orsuspected of containing an object for microscopic analysis may beintroduced into a sample holder, such as a slide or a cuvette. Thesample holder containing a sample may be introduced into a deviceconfigured to perform quantitative microscopy on the sample. Themicroscope may be coupled with an image sensor to capture imagesgenerated through the microscope objective. In the device, multipleimages of the sample may be acquired by microscopy. Any one or more ofquantitative fluorescence microscopy, quantitative dark fieldmicroscopy, quantitative bright field microscopy, and quantitative phasecontrast microscopy may be used to obtain images of the sample.Optionally, images of the entire sample in the sample holder may beacquired by microscopy. Multiple fields of view of the microscope may berequired capture images of the entire sample in the sample holder. Thesample holder may move relative to the microscope or the microscope maymove relative to the sample holder in order to generate different fieldof views in order to examine different portions of the sample in thesample holder. Multiple images of the same field of view of the samplein the sample holder may be acquired. Optionally, multiple filters maybe used with the same type of microscopy and the same field of view ofthe sample, in order to acquire different images of the same samplewhich contain different information relating to the sample. Filters thatmay be used include, without limitation band-pass and long pass filters.Filters may permit the passage of certain wavelengths of light, andblock the passage of others. Optionally, multiple types of microscopy(e.g. fluorescence, dark field, bright field, etc.) may be used toacquire images of the same field of view of the sample, in order toacquire different images of the same sample which contain differentinformation relating to the sample. Optionally, video may be used tocollect microscopy images. Optionally, microscopy images may becollected in 3-D. For microscopy performed as described herein, thedevice or system may be configured to link information relating to acell in one image of the sample to the same cell in a different image ofthe sample. Based on different images of the same sample and/or samecells, multiple attributes of cells in the sample may be determined. Insome aspects, the combination of multiple attributes/multiple pieces ofinformation about cells in a sample may be used to reach a clinicaldecision and/or to draw a conclusion about the cells that would not bepossible based on information from only a single attribute of the cells.

In some embodiments, devices and systems are provided for thequantitative measurement of two, three, four, five, or more attributesof cells in a biological sample by microscopy. In some embodiments, thedevice or system contains both a microscope or cytometer and aspectrophotometer. The device or system may further contain a fluidhandling apparatus, which is configured to move sample between aspectrophotometer and a microscope or cytometer. In some embodiments,devices and systems for performing the methods disclosed herein areconfigured as described in U.S. patent application Ser. No. 13/244,947,which is hereby incorporated by reference in its entirety. Although theforegoing has been described in the context of a cell, it should also beunderstood that some or all of the foregoing may also be applied tocrystals or other cell-sized objects that may be found in a sample.

Dynamic Dilution

In some embodiments, methods, systems, and devices are provided hereinfor dynamic dilution of cell-containing samples.

By way of non-limiting example, a method for dynamic dilution of asample may include one or more of the following steps or elements suchthat a desired number or concentration of cells or objects in the sampleis determined and this information is used as a factor in adjustingdownstream sample processing. In this non-limiting example, one or morestains may be added to a biological sample containing cells. The mixtureof stain and sample may be incubated. The cells in the mixture of stainand sample may be washed to remove excess (unbound) stain. The stained,washed cells may be prepared in a desired volume for further analysis.The stained, washed cells may be analyzed to determine the approximatenumber or concentration of cells in the sample or a portion thereof.Based on the number or concentration of stained cells in the sample orportion thereof, a volume of sample may be obtained for furtheranalysis, such that a desired number or concentration of cells forfurther analysis is obtained. In some embodiments, samples may bediluted as described in U.S. patent application Ser. No. 13/355,458,which is hereby incorporated by reference in its entirety.

In one embodiment as described herein, it is desirable to provideanother detection technique such as but not limited tofluorescence-based method for enumerating cells, to estimate cellconcentration in place of using a cell counter. This estimate isdescribe because, for accurate and reproducible staining of patientsamples, it is often desirable that stains (DNAdyes/antibodies/binders/etc.) are optimally titered for a specificnumber/concentration of cells. For example, a known concentration ofstain will be applied to a specific number of cells (e.g. 0.2 microgramsof stain per one thousand WBCs). After an incubation period, the samplewill be washed to remove excess (unbound) dye, prepared at theappropriate cell density, and imaged.

In this non-limiting example, to make an estimate of cell concentrationfor a targeted cell type, a sample is non-destructively measured with adifferent modality from that used for cytometry, such as but not limitedto a spectrophotometer, in order to inform sample processing for thecytometric assay. The method may comprise selecting another markerunique to the cell population of interest. In one non-limiting example,for B-cells, one may choose CD20. The process comprises labeling thesample with anti-CD20 binders conjugated to a different coloredfluorophore than CD5. One then measures the fluorescent signal of thissample non-destructively and rapidly using a device such as but notlimited to a fluorescence spectrophotometer. Using calibration, it ispossible to predict the concentration of B-cells with limited accuracyto provide the estimate. In one non-limiting example, the calibrationmay correlate signal strength with the number of cells for that type ofsignal. The creation of these calibration curves can be used to estimatethe number of cells or object. Other techniques for estimating number ofcells based on an overall signal strength such as but not limited tooptical, electrical, or the like are not excluded. Based on theapproximate concentration of B-cells, the system can estimate theappropriate amount and concentration of anti-CD5 binder so thatproportional relationship between CD5 expression and CD5 fluorescence ismaintained. In this manner, the stain and staining procedure can beoptimized/normalized for a particular cell number.

To maximize the use of patient samples (finger stick blood, equal orless than 120 uL), it is desirable to develop methods whereby the numberof WBCs contained within a given volume of blood, can be enumerated(i.e. WBCs/uL). This allows the number of WBCs to be determined prior toadding stains. Once determined, an exact number of cells can bealiquoted for incubation with a known concentration of stain(s),yielding optimal resolution of cell subpopulations.

Example 1—this example comprises determining the ploidy of cells(enumerate cells via fluorophore—conjugated antibody staining) In thisnon-limiting example, it is desired to enumerate the WBCs in a bloodsample so that a specific number of WBCs can be stained with apredetermined concentration of DNA dye (i.e. DAPI or propidium iodide).The method comprises counting WBCs using a fluorophore—conjugatedantibody and a spectrophotometer (similar to the dynamic dilutionperformed in the cytometry/CBC assay for WBCs). It should be understoodthat this approach may be helpful when staining cells with a DNA dye anddetermining ploidy, where the ratio of cell number to DNA dyeconcentration (cell#: [DNA dye]) is desirable for generating comparableand consistent data. Given that the number of cells per microliter ofblood vary within a healthy population, it is typically desirable todetermine the number of WBCs per microliter before attempting to stainfor ploidy.

In one embodiment, the procedure comprises using cells, where they willfirst be stained with a fluorophore-conjugated antibody (i.e. aubiquitously expressed, such as CD45, or a subpopulation specificantibody, such as CD3 for T cells) that is spectrally distinct/distantfrom the emission of the DNA dye. After an incubation period, the samplewill be washed to remove excess (unbound) antibody, prepared in theappropriate volume, and analyzed via a spectrophotometer. The resultingdata will allow for the WBCs to be enumerated/determined, so that aspecific volume of blood can be aliquoted (yielding a particular/desirednumber of WBCs) and stained with a DNA dye.

Example 2—this example comprises determining the number of cells (viaDNA staining) prior to surface staining. Additional details may also befound in the cell enumeration section herein below. It is sometimesdesirable to enumerate the WBCs in a blood sample so that a specificnumber of WBCs can be stained with optimal concentrations of antibodies.In one embodiment, the method comprises counting WBCs using a DNA dyeand a spectrophotometer (similar to the dynamic dilution performed inthe cytometry/CBC assay for WBCs).

Alternatively, if the number of cells per microliter was determinedprior to staining, then a known number of cells could be aliquoted andstained for each sample, regardless of (i) variation within a healthypopulation and (ii) disease state. To determine the number of cells permicroliter of blood, it may be possible to use DNA dyes such as DRAQ5 orpropidium iodide. After washing away the unbound dye, aspectrophotometer can be used to determine the number of nucleated(DRAQ5+) cells per microliter of blood.

In one non-limiting example, the procedure uses cells wherein the cellswill first be stained with a DNA dye (ie DRAQ5 or propidium iodide) thatis spectrally distinct/distant from the emission of thefluorophore-conjugated antibodies that will be used. After an incubationperiod, the sample will be washed to remove excess (unbound) DNA dye,prepared in the appropriate volume, and imaged via a spectrophotometer.The resulting data will allow for the WBCs to be enumerated/determined,so that a specific volume of blood can be aliquoted (yielding aparticular/desired number of WBCs) and stained with antibodies titratedfor the particular/desired number of WBCs.

Dynamic Staining

In some embodiments, methods, systems, and devices are provided hereinfor dynamic staining of cell-containing samples.

Measurement of a Component of Interest in Cells of a Cellular Population

In one embodiment, a method for dynamically staining a cell samplerelates to a method for the measurement of a component of interest incells of a cellular population in a sample.

As used herein, a “component of interest” refers to any type of moleculethat may be present in a cell. “Components of interest” includeproteins, carbohydrates, and nucleic acids. Typically, a “component ofinterest” is a specific species of molecule, such as a particularantigen. Non-limiting examples of “components of interest” of a cellinclude: CD5 protein, CD3 protein, etc.

As used herein, a “cellular population” refers to any grouping of cells,based on one or more common characteristics. A “cellular population” mayhave any degree of breadth, and may include a large number of cells oronly a small number of cells. Non-limiting examples of “cellularpopulations” include: red blood cells (RBCs), white blood cells,B-cells, CD34+ B-cells, etc.

In some circumstances, it may be desirable to quantitatively measure acomponent of interest in cells of a certain cellular population in asample from a subject. For example, it may be desirable to measure theextent of CD5 (the “component of interest”) expression in B-cells (the“cellular population”) in a sample of cells from a subject havingchronic lymphocytic leukemia. Detection and/or measurement of the levelof a component of interest may involve use of a binder molecule that hasaffinity for the specific component of interest, such an antibody orsingle chain variable fragment (“scFv”). In order to accurately measurethe level of a specific component of interest in cells in a methodinvolving the use of a binder molecule, it may be advantageous to exposethe cells to the binder molecule at a specific ratio or range of ratiosof binder molecule to target component of interest. For example, it maybe desirable to provide an amount of binder to a collection of cellssuch that there is a linear relationship between the amount of componentof interest in the cells and the amount of binder which binds to thecomponent of interest in the cells. For example, it may be undesirableto have too little binder (such that there is not enough binder to bindto all of the components of interest in the cells) or to have too muchbinder (such that the binder binds non-specifically to the cells).

Using traditional methods, it may be difficult to provide an appropriatelevel of binder to a sample in order to accurately measure the amount ofcomponent of interest in a cellular population in the sample, due to thefact that the size of the cellular population and/or component ofinterest in the sample may vary significantly between different samples.In contrast, provided herein are methods, devices, and systems fordynamically staining cell samples to accommodate samples containing awide range of cellular populations and components of interest.

In one embodiment, a method for the measurement of a component ofinterest in cells of a cellular population in a sample is provided. Themethod is not limited to but may include one or more of the followingsteps.

First, a quantitative or semi-quantitative measurement of a markerpresent in cells of the cellular population may be obtained. The markermay be any marker which is present in the cellular population ofinterest, and it may be a marker exclusively present in the cellularpopulation of interest (i.e. not present in any other cell types in thesample). Measurement of the marker may be by any method, provided themethod does not destroy the sample, and may use any system or device. Abinder which recognizes the marker may be mixed with the sample. Thebinder may have a molecule attached to facilitate detection of thebinder (e.g. a fluorescent marker). In an example, the marker may bedetected and/or measured by fluorescence spectrophotometry. Inembodiments in which the binder has a fluorescent label and the markeris measured by fluorescence spectrophotometry, fluorescencespectrophotometry may be used to measure a bulk fluorescence from thesample or a portion thereof, rather than to measure fluorescence fromindividual cells.

Second, based on the quantitative or semi-quantitative measurement ofthe marker present in cells of the cellular population, an approximateamount or concentration of cells of the cellular population present inthe sample may be determined. The approximate number or concentration ofcells in the cellular population present in the sample may bedetermined, for example, through the use of a calibration curve.Calibration curves may be prepared and/or may be available for differentmarkers/binder combinations. Calibration curves may be developed, forexample, by measuring the signal from known numbers of cells having acertain marker and bound with a certain binder. In some embodiments, theapproximate amount or concentration of cells of the cellular populationpresent in the sample may be determined with the aid of a computer. Insome aspects, the approximate number or concentration of cells in thecellular population present in the sample may be determined at no morethan about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,400, or 500% off the true concentration.

Third, based on the determined amount or concentration of cells in thecellular population present in the sample, an amount of a reagent to addto the sample may be selected, wherein the reagent binds specifically tothe component of interest in cells of the cellular population. Thereagent may be any molecule that binds specifically to the component ofinterest. For example, the reagent may be a binder, such as an antibody.The reagent may be configured such that it may be readily detected (e.g.by fluorescence or luminescence) and/or such that under at least somecircumstances, it produces a detectable signal. In some embodiments, thereagent may be attached to a molecule to facilitate detection of thereagent. The amount of reagent added to the sample may be any amount. Insome embodiments, an amount of reagent may be added to the sample suchthat there is an approximately linear relationship between the level ofthe component of interest in individual cells of the cellular populationand the signal produced by the reagents bound to the components ofinterest in individual cells of the cellular population.

Fourth, after the amount of a reagent to add to the sample is selected,the selected amount of reagent may be added to the sample.

Fifth, cells in the sample may be assayed for reagent bound to thecompound of interest.

Sixth, based on the amount of reagent bound to the component ofinterest, the amount of the component of interest in cells of thecellular population of the sample may be determined.

In some embodiments, the fifth and sixth steps may be performed togethersuch that the measurement of the amount of reagent bound to thecomponent of interest is sufficient to identify the amount of thecomponent of interest in cells of the cellular population of the sample.

In other embodiments, provided herein are systems and devices for thedynamic staining of samples. The systems and devices may contain,without limitation, a spectrophotometer and a fluorescence microscope.In an embodiment, a system or method for dynamic staining of samples maybe configured as described in U.S. patent application Ser. No.13/244,947 or 13/355,458, which are hereby incorporated by reference intheir entirety. In an embodiment, the systems and devices may beautomated to determine an amount of a reagent to add to a sample todetermine the amount of a component of interest in cells of a cellularpopulation in a sample, based on a measurement of an amount of a markerpresent in cells of the cellular population. In another embodiment, thesystems and devices may be automated to determine an amount of a reagentto add to a sample to determine the amount of a first component in cellsof a cellular population in a sample, based on a measurement of anamount of a second component in the cells of the cellular population ina sample.

Context-Based Autofocus

In some embodiments, methods, systems, and devices are provided hereinfor context-based microscopy autofocus.

The length of many clinically relevant objects in biological samplesspans a wide range. For example, bacteria are commonly about 1 μm inlength, erythrocytes are commonly about 6-8 μm in length, leukocytes arecommonly about μm 10-12 in length, epithelial cells may be about 100 μmin length, and cast and crystals may be about 200-300 μm in length. Inaddition, there are many amorphous elements such as urinary mucus whichexist as strands or filaments which may range from about 10-400 μm inlength.

A challenge in microscopy is to acquire precise images of fields of viewthat contain an unknown and arbitrary composition of objects of varioussizes, such as those described above. Since the depth of focus of manymicroscopy objectives is limited (typically about 1-10 μm), for a givenfield of view containing elements of various sizes, multiple focalplanes for the given field of view may need to be acquired in order toobtain accurate sharp images of the various elements within the field ofview. A problem with many traditional autofocus methods is that they aredesigned to focus on the dominant feature in a field of view, so thatthe sharpness of that feature can be maximized. Such methods may beineffective for capturing elements of various sizes in a sample.

In one embodiment, a method is provided for context-based microscopyautofocus, which includes mixing a reference particle of a known sizewith a sample for microscopy. The reference particle may be detectedduring microscopy, and used to achieve focusing. By use of the referenceparticles to achieve focusing, focal planes may be selected independentfrom the overall image composition. In one aspect, the method may beuseful to achieve focusing on a sample having an unknown composition ofelements. In another aspect, the method may support the generation ofprecise planes of focus, independent of the precision of the microscopeor microscopy-related hardware. For example, when a plane of focus isselected based on feedback from the sharpness of the reference particleswithin a field of view, precise focusing on various elements within asample may be achieved, regardless of the level of accuracy or precisionof the focusing hardware [e.g. the microscope objective actuation, theshape of a sample holder (e.g. a cuvette or slide), or thenon-uniformity of a sample holder].

In an embodiment, a reference particle may contain or be labeled with amolecule to facilitate detection of the particle during microscopy. Inone example, a reference particle may be labeled with or contain afluorescent molecule. The fluorescent molecule may absorb light at afirst wavelength of light, and, in response to the absorbance of thefirst wavelength of light, it may emit light at a second wavelength. Inan embodiment, a sample mixed with a reference particle may be exposedto a wavelength of light capable of exciting a fluorescent molecule in areference particle of interest and emitted light from the fluorescentmolecule may be measured. Specific fluorescence from a referenceparticle may be used to detect reference particles, and information fromdetected reference particles in a sample may be used for autofocusing.

Reference particles may be of any shape, such as spherical or cuboid.Reference particles include, without limitation, beads and microspheres.Reference particles may be of any size, such as with a diameter orlength of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 μm. Referenceparticles may contain any material, such as polystyrene, polystyrene,latex, acrylic, or glass.

In one embodiment, a method for focusing a microscope is provided, whichmay include one or more of the following steps. First, a samplecontaining an object for microscopic analysis (e.g. bacteria,erythrocytes, etc.) may be mixed with a reference particle. Thereference particle may contain or be labeled with a molecule tofacilitate the detection of the particle, such as a fluorophore. Second,the mixture containing the reference particle and the sample may bepositioned into a light path of a microscope, for example in cuvette orslide. Optionally, the reference particle may sink to the bottom of thesample in the cuvette or slide, such that the reference particle restson the lowest surface of the cuvette or slide which is in contact withthe sample. The microscope may be of any type, including a fluorescentmicroscope. Third, the mixture may be exposed to a light beam configuredto visualize the reference particle. The light beam may be of any type,and may be of any orientation relative to the reference particle. Forexample, the light beam may be at a wavelength capable of exciting afluorophore within or attached to the reference particle. Exposure ofthe reference particle to the light beam may result in, for example, thegeneration and emission of light at a particular wavelength from thereference particle and/or scattering of light from the referenceparticle. Fourth, light emitted or scattered from the reference particlemay be detected by the microscope, and this information may be used inorder to determine the position of the reference particle within themixture and/or to focus the microscope. Optionally, the microscope maybe focused into a plane of focus suited for objects of similar size tothe reference particle. An image from the microscope may be obtained byan image sensor. The image may be saved and/or or used for imageanalysis.

In some embodiments, a plurality of reference particles may be added toa sample. The reference particles may be all of the same size, or theymay be of different sizes. In some embodiments, reference particles ofdifferent sizes contain different fluorophores. Different fluorophoresmay have different absorption wavelengths, different emissionwavelengths, or both.

In an embodiment, a method for focusing a microscope is provided,including mixing more than one reference particle of known size with asample for microscopy, wherein at least two of the reference particlesare of different sizes and contain different fluorophores. The methodmay include one or more of the following steps. First, a samplecontaining an object for microscopic analysis may be mixed with two ormore reference particles, wherein at least two of the referenceparticles are of different sizes and contain different fluorophores(i.e. the “first reference particle” and the “second referenceparticle”). Second, the mixture containing the reference particles andthe sample may be positioned into the light path of a microscope. Themicroscope may be of any type, including a fluorescent microscope.Third, the mixture may be exposed to a light beam configured tovisualize the first reference particle. The light beam may be of anytype, and may be of any orientation relative to the first referenceparticle. For example, the light beam may be at a wavelength capable ofexciting a fluorophore within or attached to the first referenceparticle. Exposure of the first reference particle to the light beam mayresult in the generation and emission or scattering of light at aparticular wavelength from the first reference particle. Fourth, lightemitted or scattered from the first reference particle may be detected,and this information may be used in order to determine the position ofthe first reference particle within the mixture and/or to focus themicroscope into a first plane of focus suited for objects of similarsize to the first reference particle. Optionally, an image of the firstfocal plane may be obtained by an image sensor. The image may be savedand/or or used for image analysis. Fifth, the mixture may be exposed toa light beam configured to visualize the second reference particle. Thelight beam may be of any type, and may be of any orientation relative tothe second reference particle. Exposure of the second reference particleto the light beam may result in the generation and emission orscattering of light at a particular wavelength from the second referenceparticle. Sixth, light emitted or scattered from the second referenceparticle may be detected, and this information may be used in order todetermine the position of the second reference particle within themixture and/or to focus the microscope into a second plane of focussuited for objects of similar size to the second reference particle.Optionally, an image of the second focal plane may be obtained by animage sensor. The image may be saved and/or or used for image analysis.

In other embodiments, provided herein are systems and devices forcontext-based microscopy autofocus. The systems and devices may contain,without limitation, a fluorescence microscope. In an embodiment, thesystems and devices may be automated to add a reference particle havinga known size to a sample for microscopic analysis to form a mixture, toposition the mixture into the light path of a microscope, to expose themixture to a light beam configured to visualize the reference particle,to determine the position of the reference particle within the mixtureand/or to focus the microscope based on the position of the referenceparticle within the mixture. In an embodiment, a system or method forcontext-based microscopy autofocus may be configured as described inU.S. patent application Ser. No. 13/244,947 or 13/355,458, which arehereby incorporated by reference in their entirety.

Cell Counting/Enumerating Cells

In some embodiments, methods, systems, and devices are provided hereinfor enumerating cells in a sample.

Certain traditional methods for staining cell-containing samples involvestaining a specific volume of a sample (e.g. blood) with a particularconcentration or amount of stain. This may be referred to as “volumetricstaining” Volumetric staining has a number of shortcomings, including:(i) it fails to address normal variations in cell subpopulations betweendifferent subjects (e.g. different healthy subjects may have widelydifferent numbers of subpopulations of cells, such as CD3+ T cells) and(ii) it fails to address that pathological samples may have dramaticallydifferent cellular composition when compared to healthy samples (e.g.the percent and number of CD3+ T cells in blood are greatly elevated inpatients with T cell leukemia over the percent and number in healthysubjects).

For accurate and reproducible staining of cell-containing samples, itmay be desirable to add a specific amount of a cellular stain (e.g. DNAdyes, antibodies, binders, etc.) to a specific number or concentrationof cells. For example, it may be desirable to add 0.2 micrograms of aparticular stain for white blood cells per 1000 white blood cells in asample. After an incubation period of the dye with the cells, a samplemay be washed to remove excess (unbound) dye, prepared to an appropriatecell density for microscopy, and imaged. In this manner, a stain andstaining procedure can be optimized or normalized for a particular cellnumber.

In one embodiment, a method is provided for enumerating the number ofcells in a sample. The method may include one or more of the followingsteps or elements. A first stain that will bind to the cells of interestin a sample may be added to the sample. The mixture of first stain andsample may be incubated. The cells in the mixture of first stain andsample may be washed to remove excess (unbound) stain. The washed cellsstained with a first stain may be prepared in a desired volume forfurther analysis. The washed cells stained with a first stain may beanalyzed by a spectrophotometer. Data from the spectrophotometer may beused to enumerate the approximate number of cells in the sample. Basedon the number of cells in the sample, a second stain that will bind tocells of interest in a sample may be added to the sample. The mixture ofsecond stain and sample may be incubated. The cells in the mixture ofsecond stain and sample may be washed to remove excess stain. The washedcells stained with a second stain may be prepared in a desired volumefor further analysis. The washed cells stained with a second stain maybe analyzed by microscopy.

Enumerating Cells in a Sample Prior to Determining the Ploidy of Cells

In one embodiment, a method for enumerating cells in a sample prior todetermining the ploidy of the cells is provided, wherein the methodincludes one or more of the following steps or elements. A first stainwhich binds to the cells of interest in the sample and that isspectrally distinct from the emission of a DNA dye may be added to thesample. The cells of interest may be, for example, white blood cells.The first stain may be, for example, a fluorphore-conjugated antibody. Afluorphore-conjugated antibody may bind to, for example, a widelyexpressed antigen (e.g. CD45), or it may bind to an antigen expressed bya specific sub-population of cells (e.g. CD3 for T cells). The mixtureof first stain and sample may be incubated. The cells in the mixture offirst stain and sample may be washed to remove excess (unbound) stain.The washed cells stained with a first stain may be prepared in a desiredvolume for further analysis. The washed cells stained with a first stainmay be analyzed by a spectrophotometer. Data from the spectrophotometermay be used to enumerate the approximate number of cells in the sample.Based on the number of cells in the sample, a second stain that willbind to cells of interest in a sample may be added to the sample. Thesecond stain may be a DNA dye, such as propidium iodide or4′,6-diamidino-2-phenylindole (“DAPI”). The mixture of second stain andsample may be incubated. The cells in the mixture of second stain andsample may be washed to remove excess stain. The washed cells stainedwith a second stain may be prepared in a desired volume for furtheranalysis. The washed cells stained with a second stain may be analyzedfor ploidy by microscopy.

In methods for determining the ploidy of cells, it may be important tocombine a given number of cells for ploidy analysis with a certainamount or concentration of DNA stain, in order to generate accurate andconsistent data regarding the ploidy of the cells. In one example, thenumber of white blood cells per volume of blood may vary within ahealthy population, and thus, it may be desirable to determine thenumber of white blood cells in a volume of blood before attempting tostain the white blood cells for ploidy analysis.

The methods provided above for determining the ploidy of cells may alsobe performed for any method in which enumerating cells in a sample priorto determining an attribute related to the nucleic acid content of acell is desired. For example, the above method may be used with methodsinvolving enumerating cells in a sample prior to determining themorphology of nuclei of cells, the size of the nuclei of cells, theratio of nuclei area to total cell area, etc.

Enumerating Cells in a Sample Prior to Cell Surface Staining

In one embodiment, a method for enumerating cells in a sample prior tocell surface staining is provided, wherein the method includes one ormore of the following steps or elements. A first stain which binds tothe cells of interest in the sample and that is spectrally distinct fromthe emission of a dye to be used to stain the surface of the cells ofinterest may be added to the sample. The cells of interest may be, forexample, white blood cells. The first stain may be, for example, a DNAdye (e.g. propidium iodide or DAPI). The mixture of first stain andsample may be incubated. The cells in the mixture of first stain andsample may be washed to remove excess (unbound) stain. The washed cellsstained with a first stain may be prepared in a desired volume forfurther analysis. The washed cells stained with a first stain may beanalyzed by a spectrophotometer. Data from the spectrophotometer may beused to enumerate the approximate number of cells in the sample. Basedon the number of cells in the sample, a second stain that will bind tocells of interest in a sample may be added to the sample. The secondstain may be, for example, a fluorphore-conjugated antibody. Afluorphore-conjugated antibody may bind to, for example, a widelyexpressed antigen (e.g. CD45), or it may bind to an antigen expressed bya specific sub-population of cells (e.g. CD3 for T cells). The mixtureof second stain and sample may be incubated. The cells in the mixture ofsecond stain and sample may be washed to remove excess stain. The washedcells stained with a second stain may be prepared in a desired volumefor further analysis. The washed cells stained with a second stain maybe analyzed for a cell surface antigen by microscopy.

In methods for cell surface antigen staining of cells, it may beimportant to combine a given number of cells for analysis with a certainamount or concentration of cell surface antigen stain, in order togenerate accurate and consistent data regarding the content of the cellsurfaces. In one example, the number of white blood cells per volume ofblood may vary within a healthy population, and thus, it may bedesirable to determine the number of white blood cells in a volume ofblood before attempting to stain the white blood cells for cell surfaceantigens. In another example, the number of white blood cells per volumeof blood may vary between healthy and sick subjects, and thus, it may bedesirable to determine the number of white blood cells in a volume ofblood before attempting to stain the white blood cells for cell surfaceantigens. As a theoretical example, a healthy patient may have 100 cellsper microliter of blood, and 10 of these are CD3+ T cells, while alymphoma patient may have 1000 cells per microliter of blood and 900 ofthese are CD3+ T cells. If 100 microliters of blood is traditionallystained, then a sample from a healthy subject would contain 10,000 totalcells/1000 CD3+ T cells, and a sample from a lymphoma subject wouldcontain 100,000 total cells/90,000 CD3+ T cells. In this theoreticalexample, the pathological sample contains ten times the number of totalcells and ninety times the number of CD3+ T cells, when compared to asample from a healthy subject. If the pathological sample would bestained with a traditional “volumetric staining” approach that isoptimized for samples from healthy subjects, the sample from thelymphoma subject may be insufficiently stained.

Accordingly, methods provided herein may be used to enumerate cells in asample before cell staining, in order to generate accurate and/orconsistent data regarding samples.

Method Speeds

Methods, systems, and devices provided herein may support the rapiddevelopment of sample analysis results. Any of the methods providedherein may provide analysis results in less than about 6 hours, 4 hours,3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10minutes, or 5 minutes from the initiation of the method.

Rapid analysis results may be used to provide real-time informationrelevant to the treatment, diagnosis, or monitoring of a patient. Forexample, rapid analysis results may be used to guide a treatmentdecision of a surgeon operating on a patient. During surgery, a surgeonmay obtain a biological sample from a patient for analysis. By receivingrapid analysis of a sample by a method provided herein, a surgeon may beable to make a treatment decision during the course of surgery.

In another example, rapid analysis results provided by the methods,systems, and devices provided herein may support a patient receivinginformation regarding a biological sample provided by the patient at apoint of service during the same visit to the point of service locationin which the patient provided the biological sample.

Analysis of Pathology Samples

Any of the methods provided herein may be used to analyzecell-containing pathology samples. If a pathology sample is a tissuesample, the sample may be treated to separate the cells of the tissueinto individual cells for analysis by methods provided herein.

Analysis of pathology samples by any of the methods provided herein maysupport rapid pathology analysis, and the rapid integration of pathologyanalysis results into a treatment decision for a patient.

Additional Procedures in Response to Analysis Results

In some embodiments, the devices and systems provided herein may beconfigured to trigger an additional procedure in response to a resultobtained by an analysis method provided herein.

In one example, a device or system may be programmed to provide an alertto a user if a result is outside of an expected range. The alert mayprompt a user or medical personnel to, for example, manually analyze asample, check the device or system for proper operation, etc.

In another example, a device or system may be programmed toautomatically run one or more additional tests on a sample if a resultis within or outside of a certain range. In some examples, devices andsystems provided herein are capable of performing multiple differentassays, and the device or system may run an addition assay to verify orfurther investigate a result generated by a method provided herein.

Analysis Using Non-Specific Dyes

One non-limiting example to accelerate imaging is to use a “high light”situation, where cells are labeled with very high concentration of dyes.In the present embodiment, non-specific dyes are used that label theDNA, the membranes, or other portion of the cells. This example does notuse anti-body specific dyes.

With the non-specific dye, it is possible to obtain cell informationwithout centrifugation or performing physical separation. Without thisseparation step, one can more rapidly move directly to imaging thesample, such as but not limited imaging a large area of cells that mayinclude both a) non-target cells such as red blood cells (RBCs) and b)target cells or objects of interest such as white blood cells (WBCs).Thus, in one non-limiting example, one can image five million RBCs andfive thousand or other number of WBCs therein. The targeted cells can bedifferentiated based on what is inside the cell such as but not limitedto the shape of nucleus of cell. In one embodiment, this stained thenucleus and based on what a cell has, one can determine cell type basedon this, even though the dye is non-specific. In other examples, otherinternal shapes in the cell such as cytoplasm has granules or otherobjects therein. For urine sample, it is the cells and crystal shapesthat can be used. In this manner, the use of non-specific dyes can beused to rapidly image cells in a manner that can be used to determinecells as desired.

Analysis Using a Plurality of Excitation and/or Detection Channels

In the context of using even smaller sample volumes for cytometry, onething for advanced cytometry assays is to use additional excitationand/or detection wavelengths. For example, for classification of thelymphocyte subset assay, the various cells such as T cells, B cells, Kcells etc. . . . are to be counted. In this case, just to identify thatthe cell is a lymphocyte, one uses two markers. To further sub-classify,one use two markers. If one has a system that can only detect two colorsat a time, there is an insufficient number of wavelengths.

In one embodiment, one can aliquot the sample to make two separatesamples and image one combination in one part and another combination inanother part. Unfortunately, this can cause a doubling of volume andtime. The more independent channels one build into our system, thelesser is the number of these sample parts or volume is used.

EXAMPLES

Cell Processing

In embodiments, it is often useful to process biological samples forimaging, testing, and analysis. For example, it is often useful toprocess biological samples containing cells for imaging, testing, andanalysis.

Processing of a biological sample may include pre-processing (e.g.,preparation of a sample for a subsequent processing or measurement),processing (e.g., alteration of a sample so that it differs from itsoriginal, or previous, state), and post-processing (e.g., disposal ofall or a portion of a sample following its measurement or use). Abiological sample may be divided into portions, such as aliquots of ablood or urine sample, or such as slicing, mincing, or dividing a tissuesample into two or more pieces. Processing of a biological sample, suchas blood sample, may include mixing, stirring, sonication,homogenization, or other processing of a sample or of a portion of thesample. Processing of a biological sample, such as blood sample, mayinclude centrifugation of a sample or a portion thereof. Processing of abiological sample, such as blood sample, may include providing time forcomponents of the sample to separate or settle, and may includefiltration (e.g., passing the sample or a portion thereof through afilter). Processing of a biological sample, such as blood sample, mayinclude allowing or causing a blood sample to coagulate. Processing of abiological sample, such as blood sample, may include concentration ofthe sample, or of a portion of the sample (e.g., by sedimentation orcentrifugation of a blood sample, or of a solution containing ahomogenate of tissue from a tissue sample) to provide a pellet and asupernatant. Processing of a biological sample, such as blood sample,may include dilution of a portion of the sample. Dilution may be of asample, or of a portion of a sample, including dilution of a pellet orof a supernatant from sample. A biological sample may be diluted withwater, or with a saline solution, such as a buffered saline solution. Abiological sample may be diluted with a solution which may or may notinclude a fixative (e.g., formaldehyde, paraformaldehyde, or other agentwhich cross-links proteins). A biological sample may be diluted with asolution effective that an osmotic gradient is produced between thesurrounding solution and the interior, or an interior compartment, ofsuch cells, effective that the cell volume is altered. For example,where the resulting solution concentration following dilution is lessthan the effective concentration of the interior of a cell, or of aninterior cell compartment, the volume of such a cell will increase(i.e., the cell will swell). A biological sample may be diluted with asolution which may or may not include an osmoticant (such as, forexample, glucose, sucrose, or other sugar; salts such as sodium,potassium, ammonium, or other salt; or other osmotically active compoundor ingredient). In embodiments, an osmoticant may be effective tomaintain the integrity of cells in the sample, by, for example,stabilizing or reducing possible osmotic gradients between thesurrounding solution and the interior, or an interior compartment, ofsuch cells. In embodiments, an osmoticant may be effective to provide orto increase osmotic gradients between the surrounding solution and theinterior, or an interior compartment, of such cells, effective that thecells at least partially collapse (where the cellular interior or aninterior compartment is less concentrated than the surroundingsolution), or effective that the cells swell (where the cellularinterior or an interior compartment is more concentrated than thesurrounding solution).

A biological sample may be dyed, or markers may be added to the sample,or the sample may be otherwise prepared for detection, visualization, orquantification of the sample, a portion of a sample, a component part ofa sample, or a portion of a cell or structure within a sample. Forexample, a biological sample may be contacted with a solution containinga dye. A dye may stain or otherwise make visible a cell, or a portion ofa cell, or a material or molecule associated with a cell in a sample. Adye may bind to or be altered by an element, compound, or othercomponent of a sample; for example a dye may change color, or otherwisealter one of more of its properties, including its optical properties,in response to a change or differential in the pH of a solution in whichit is present; a dye may change color, or otherwise alter one of more ofits properties, including its optical properties, in response to achange or differential in the concentration of an element or compound(e.g., sodium, calcium, CO2, glucose, or other ion, element, orcompound) present in a solution in which the dye is present. Forexample, a biological sample may be contacted with a solution containingan antibody or an antibody fragment. For example, a biological samplemay be contacted with a solution that includes particles. Particlesadded to a biological sample may serve as standards (e.g., may serve assize standards, where the size or size distribution of the particles isknown, or as concentration standards, where the number, amount, orconcentration of the particles is known), or may serve as markers (e.g.,where the particles bind or adhere to particular cells or types ofcells, to particular cell markers or cellular compartments, or where theparticles bind to all cells in a sample).

Cytometry includes observations and measurements of cells, such as redblood cells, platelets, white blood cells, including qualitative andquantitative observations and measurements of cell numbers, cell types,cell surface markers, internal cellular markers, and othercharacteristics of cells of interest. Where a biological sample includesor is a blood sample, the sample may be divided into portions, and maybe diluted (e.g., to provide greater volume for ease of handling, toalter the density or concentration of cellular components in the sampleto provide a desired diluted density, concentration, or cell number orrange of these, etc.). The sample may be treated with agents whichaffect coagulation, or may be treated or handled so as to concentrate orprecipitate sample components (e.g., ethylene diamine tetraacetic acid(EDTA) or heparin may be added to the sample, or the sample may becentrifuged or cells allowed to settle). A sample, or portion of asample, may be treated by adding dyes or other reagents which may reactwith and mark particular cells or particular cellular components. Forexample, dyes which mark cell nuclei (e.g., hematoxylin dyes, cyaninedyes, drag dyes such as Draq5, and others); dyes which mark cellcytoplasm (e.g., eosin dyes, including fluorescein dyes, and others) maybe used separately or together to aid in visualization, identification,and quantification of cells. More specific markers, including antibodiesand antibody fragments specific for cellular targets, such as cellsurface proteins, intracellular proteins and compartments, and othertargets, are also useful in cytometry.

Biological samples may be measured and analyzed by cytometry usingoptical means, including, for example, photodiode detectors,photomultipliers, charge-coupled devices, laser diodes,spectrophotometers, cameras, microscopes, or other devices which measurelight intensity (of a single wavelength, of multiple wavelengths, or ofa range, or ranges, of wavelengths of light), form an image, or both. Afield of view including a sample, or portion of a sample, may be imaged,or may be scanned, or both, using such detectors. A biological samplemay be measured and analyzed by cytometry prior to processing, dilution,separation, centrifugation, coagulation, or other alteration. Abiological sample may be measured and analyzed by cytometry during orfollowing processing, dilution, separation, centrifugation, coagulation,or other alteration of the sample. For example, a biological sample maybe measured and analyzed by cytometry directly following receipt of thesample. In other examples, a biological sample may be measured andanalyzed by cytometry during or after processing, dilution, separation,centrifugation, coagulation, or other alteration of the sample.

For example, a blood sample or portion thereof may be prepared forcytometry by sedimentation or centrifugation. A sedimented or pelletportion of such a sample may be resuspended in a buffer of choice priorto cytometric analysis (e.g., by aspiration, stirring, sonication, orother processing). A biological sample may be diluted or resuspendedwith water, or with a saline solution, such as a buffered salinesolution prior to cytometric analysis. A solution used for such dilutionor resuspension may or may not include a fixative (e.g., formaldehyde,paraformaldehyde, or other agent which cross-links proteins). A solutionused for such dilution or resuspension may provide an osmotic gradientbetween the surrounding solution and the interior, or an interiorcompartment, of cells in the sample, effective that the cell volume ofsome or all cells in the sample is altered. For example, where theresulting solution concentration following dilution is less than theeffective concentration of the interior of a cell, or of an interiorcell compartment, the volume of such a cell will increase (i.e., thecell will swell). A biological sample may be diluted with a solutionwhich may or may not include an osmoticant (such as, for example,glucose, sucrose, or other sugar; salts such as sodium, potassium,ammonium, or other salt; or other osmotically active compound oringredient). In embodiments, an osmoticant may be effective to maintainthe integrity of cells in the sample, by, for example, stabilizing orreducing possible osmotic gradients between the surrounding solution andthe interior, or an interior compartment, of such cells. In embodiments,an osmoticant may be effective to provide or to increase osmoticgradients between the surrounding solution and the interior, or aninterior compartment, of such cells, effective that the cells at leastpartially collapse (where the cellular interior or an interiorcompartment is less concentrated than the surrounding solution), oreffective that the cells swell (where the cellular interior or aninterior compartment is more concentrated than the surroundingsolution).

For example, a biological sample may be measured or analyzed followingdilution of a portion of the sample with a solution including dyes. Forexample, a biological sample may be measured or analyzed followingdilution of a portion of the sample with a solution including antibodiesor antibody fragments. For example, a biological sample may be measuredor analyzed following dilution of a portion of the sample with asolution including particles. Particles added to a biological sample mayserve as standards (e.g., may serve as size standards, where the size orsize distribution of the particles is known, or as concentrationstandards, where the number, amount, or concentration of the particlesis known), or may serve as markers (e.g., where the particles bind oradhere to particular cells or types of cells, to particular cell markersor cellular compartments, or where the particles bind to all cells in asample).

For example, a biological sample may be measured or analyzed followingprocessing which may separate one or more types of cells from anothercell type or types. Such separation may be accomplished by gravity(e.g., sedimentation); centrifugation; filtration; contact with asubstrate (e.g., a surface, such as a wall or a bead, containingantibodies, lectins, or other components which may bind or adhere to onecell type in preference to another cell type); or other means.Separation may be aided or accomplished by alteration of a cell type ortypes. For example, a solution may be added to a biological sample, suchas a blood sample, which causes some or all cells in the sample toswell. Where one type of cell swells faster than another type or typesof cell, cell types may be differentiated by observing or measuring thesample following addition of the solution. Such observations andmeasurements may be made at a time, or at multiple times, selected so asto accentuate the differences in response (e.g., size, volume, internalconcentration, or other property affected by such swelling) and so toincrease the sensitivity and accuracy of the observations andmeasurements. In some instances, a type or types of cells may burst inresponse to such swelling, allowing for improved observations andmeasurements of the remaining cell type or types in the sample.

Observation, measurement and analysis of a biological sample bycytometry may include photometric measurements, for example, using aphotodiode, a photomultiplier, a laser diode, a spectrophotometer, acharge-coupled device, a camera, a microscope, or other means or device.Cytometry may include preparing and analyzing images of cells in abiological sample (e.g., two-dimensional images), where the cells arelabeled (e.g., with fluorescent, chemiluminescent, enzymatic, or otherlabels) and plated (e.g., allowed to settle on a substrate) and imagedby a camera. The camera may include a lens, and may be attached to orused in conjunction with a microscope. Cells may be identified in thetwo-dimensional images by their attached labels (e.g., from lightemitted by the labels).

An image of cells prepared and analyzed by a cytometer as disclosedherein may include no cells, one cell, or multiple cells. A cell or cellin an image of a cytometer, as disclosed herein, may be labeled, asdisclosed above. A cell or cell in an image of a cytometer, as disclosedherein, may be labeled, as disclosed above, effective to identify theimage, and the subject from whom the sample was taken.

In some embodiments, the assay system is configured to perform cytometryassays. Cytometry assays are typically used to optically, electrically,or acoustically measure characteristics of individual cells. For thepurposes of this disclosure, “cells” may encompass non-cellular samplesthat are generally of similar sizes to individual cells, including butnot limited to vesicles (such as liposomes), small groups of cells,virions, bacteria, protozoa, crystals, bodies formed by aggregation oflipids and/or proteins, and substances bound to small particles such asbeads or microspheres. Such characteristics include but are not limitedto size; shape; granularity; light scattering pattern (or opticalindicatrix); whether the cell membrane is intact; concentration,morphology and spatio-temporal distribution of internal cell contents,including but not limited to protein content, protein modifications,nucleic acid content, nucleic acid modifications, organelle content,nucleus structure, nucleus content, internal cell structure, contents ofinternal vesicles (including pH), ion concentrations, and presence ofother small molecules such as steroids or drugs; and cell surface (bothcellular membrane and cell wall) markers including proteins, lipids,carbohydrates, and modifications thereof. By using appropriate dyes,stains, or other labeling molecules either in pure form, conjugated withother molecules or immobilized in, or bound to nano- or micro-particles,cytometry may be used to determine the presence, quantity, and/ormodifications of specific proteins, nucleic acids, lipids,carbohydrates, or other molecules. Properties that may be measured bycytometry also include measures of cellular function or activity,including but not limited to phagocytosis, antigen presentation,cytokine secretion, changes in expression of internal and surfacemolecules, binding to other molecules or cells or substrates, activetransport of small molecules, mitosis or meiosis; protein translation,gene transcription, DNA replication, DNA repair, protein secretion,apoptosis, chemotaxis, mobility, adhesion, antioxidizing activity, RNAi,protein or nucleic acid degradation, drug responses, infectiousness, andthe activity of specific pathways or enzymes. Cytometry may also be usedto determine information about a population of cells, including but notlimited to cell counts, percent of total population, and variation inthe sample population for any of the characteristics described above.The assays described herein may be used to measure one or more of theabove characteristics for each cell, which may be advantageous todetermine correlations or other relationships between differentcharacteristics. The assays described herein may also be used toindependently measure multiple populations of cells, for example bylabeling a mixed cell population with antibodies specific for differentcell lines. A microscopy module may permit the performance of histology,pathology, and/or morphological analysis with the device, and alsofacilitates the evaluation of objects based on both physical andchemical characteristics. Tissues can be homogenized, washed, depositedon a cuvette or slide, dried, stained (such as with antibodies),incubated and then imaged. When combined with the data transmissiontechnologies described elsewhere herein, these innovations facilitatethe transmission of images from a CMOS/CDD or similar detector to, e.g.,a licensed pathologist for review, which is not possible withtraditional devices that only perform flow cytometry. The cytometer canmeasure surface antigens as well as cell morphology; surface antigensenable more sensitive and specific tesing compared to traditionalhematology laboratory devices. The interpretation of cellular assays maybe automated by gating of one or more measurements; the gatingthresholds may be set by an expert and/or learned based on statisticalmethods from training data; gating rules can be specific for individualsubjects and/or populations of subjects.

In some embodiments, the incorporation of a cytometer module into apoint of service device provides the measurement of cellular attributestypically measured by common laboratory devices and laboratories forinterpretation and review by classically-trained medical personnel,improving the speed and/or quality of clinical decision-making. A pointof service device may, therefore, be configured for cytometric analysis.

Example 1

A sample of cells containing blood leukocytes including natural killercells and neutrophils was obtained. The sample was treated with afluorescently labeled identity binder (anti-CD 16 binder), which bindsto both natural killer cells and neutrophils. The sample was alsotreated with a nuclear dye (DRAQ5). The sample was imaged byfluorescence microscopy and dark field microscopy. The level offluorescence and light side scatter of different cells in the sample wasrecorded and analyzed. Segmented images containing the anti-CD16 bindersignal provided quantitative information on the fluorescence intensityof each cell (corresponding to the CD 16 expression level), and also thesize of each cell. The darkfield image provided quantitative informationon the scatter properties of each cell. Images containing the DNA dyesignal were segmented to determine the fluorescent intensity, size, andshape of the nucleus.

As shown in FIG. 1A, two major groupings cells were identified based onthe measurement of CD16 fluorescence and light scatter of the differentcells. The group of cells with bright/high CD16 fluorescence signal andhigh scatter (FIG. 1A, right circle) are neutrophils. The group of cellswith intermediate CD16 fluorescence signal and low scatter (FIG. 1A,left circle) are natural killer cells. While the measurement offluorescence and light scatter of the different cells provides enoughinformation to classify most cells in the sample as either naturalkiller cells or neutrophils, for some cells, measurement of theseattributes does not provide enough information to classify the cellswith a high degree of accuracy. For example, the measurement offluorescence and light scatter of cells does not provide enoughinformation to accurately classify the small group of cells in thesmallest circle in FIG. 1A (i.e. the middle circle). In order toidentify whether the cells in the smallest circle were natural killercells or neutrophils, images of the nuclear (DRAQ5) and total cell(anti-CD16) staining of these were examined. Quantitative measurementsof the area of the nucleus and the total cell volume of the cells wereobtained, and the ratio of nuclear area to total cell area wasdetermined. As shown in FIG. 1B, there is a clear difference in theratio of nuclear area to total cell area between natural killer cells(“NK”) and neutrophils (“Neu”). Thus, the use of quantitative microscopyto examine multiple attributes of cells in the sample was used to allowfor unambiguous classification of cells. FIG. 1C shows images of naturalkiller cells from the smallest circle in FIG. 1A. All images have thesame length scale. The images on the left are cells stained for totalcell area (anti-CD16), and the images on the right are the same cellswith just nuclear staining (DRAQ5). The images on the top and bottom roware different examples of the natural killer cells. FIG. 1D shows imagesof neutrophils from the smallest circle in FIG. 1A. All images have thesame length scale. The images on the left are cells stained for totalcell area, and the images on the right are the same cells with justnuclear staining. The images on the top and bottom row are differentexamples of the natural killer cells.

In addition, the nucleus of a neutrophil has a distinctive multi-lobedshape, whereas the nucleus of a natural killer cell (and otherlymphocytes) is round, even, and smooth. Image segmentation algorithmsmay be used to identify and classify cells based on the shape of thenucleus itself

Example 2

A sample containing platelets was obtained. The platelets were labeledwith fluorescently conjugated anti-CD41 and anti-CD61 antibodies. Beadshaving a diameter of 3 μm were also added to the sample. The sample wasimaged at 10× and 20× magnifications (FIG. 2A). The intensity offluorescence distribution for individual platelets was measured (fromboth antibodies), and determined have a Gaussian shape (FIG. 2B). Themeasured values of fluorescence of individual platelets was plotted, anda fit for the intensity distribution was determined (FIG. 2C). In FIG.2C, the grey line is the measured fluorescence intensity across anindividual platelet, and the black line is the fit. Parameters of thefit, such as the mean of the Gaussian, the variance, the volume, thewidth, and the area of the base, etc., can be evaluated as predictors ofplatelet volume. The volume of the Gaussian and the width of the fithave been determined to correlate closely with mean platelet volume.

For the above measurements, the 3 μm beads served as references andfiducials for controlling variance in accurately determining the bestplane of focus, and the effect of this variance on the measurement ofvolume.

In addition, platelet size estimated based on fitting a 2D model can becalibrated to be in the normal range (FIG. 3).

Example 3

A sample containing red blood cells (“RBCs”) was obtained. The RBCs weretreated to swell the RBCs into a sphere-like shape, by treating the RBCswith a low concentration of a surfactant (DDAPS or SDS). The RBCs wereimaged by dark field microscopy in two different cuvettes: (A) a cuvettethat allowed only pure epi-illumination (FIG. 4A); and (B) a cuvettethat allowed a mixture of both epi and trans-illumination (FIG. 4B). TheRBCs were much more visible in the cuvette that allowed a mixture ofboth epi and trans-illumination over the cuvette that allowed only pureepi-illumination (FIG. 4).

Example 4

A sample containing neutrophils was obtained. In neutrophils, the shapeand chromatin morphology of the nucleus may indicate whether it is animmature “band” neutrophil or a mature “segmented” neutrophil. Bandneutrophils are immature neutrophils that have recently emerged from thebone marrow. An increase in the proportion of band neutrophils mayindicate an ongoing infection or inflammation.

The sample was mixed with a fluorescently labeled anti-CD16 antibody,which recognizes CD16, a cell surface receptor on neutrophils. Thesample was also stained with a fluorescent nuclear dye. The sample wasimaged by fluorescence microscopy, to obtain both nuclear staining andCD16 staining data from the cells. Band neutrophils generally havesimilar expression levels of CD 16 as mature segmented neutrophils, andthus cannot be distinguished by virtue of fluorescence intensity from CD16 staining alone.

Image analysis including image segmentation is used to recognize nuclearstaining and morphologies of band neutrophils and segmented neutrophils,thereby allowing classification of the cells. The size, shape, andfluorescence intensity of the nucleus of cells are examined. Inaddition, the nuclei are analyzed to determine the number of lobes(peaks in intensity within the nuclear area), distance between the lobesof the nucleus, and the changes in curvature (second derivative) of thenuclear outline. FIG. 5A shows representative images of bandneutrophils. In these images, the nucleus appears as a light grey, andthe cell cytoplasm appears as a darker grey. As neutrophilsdifferentiate through the myeloid lineage, they develop a characteristic“U” shaped nucleus prior to reaching full maturity. FIG. 5B showsrepresentative images of segmented neutrophils. In these images, thenucleus appears as a light grey, and the cell cytoplasm appears as adarker grey. The nuclei of segmented neutrophils have multiplesegments/lobes (typically about 3-5). Thus, this analysis supportsidentification and quantification of different subpopulations ofneutrophils in the blood.

Example 5

A sample of cells from a subject with chronic lymphocytic leukemia (CLL)is obtained. The objective is to quantify the extent of CD5 expressionon B-cells from the subject. Anti-CD20 antibodies are selected as thebinder for B-cells. Anti-CD20 antibodies labeled with a first coloredfluorphore are mixed with the sample. After an appropriate incubationtime, the sample is washed and the unbound anti-CD20 antibodies areremoved. The sample is exposed to a light source capable of exciting thefirst fluorophore, and fluorescent signal is measured using aspectrophotometer. Based on the fluorescent signal, the approximateconcentration of B-cells in the sample is determined. The determinedapproximate concentration of B-cells is, in fact, within 1.5 fold of thetrue concentration of B-cells in the sample.

Based on the approximate concentration of B-cells in the sample, anappropriate amount of anti-CD5 binder is added to the sample so that aproportional relationship between CD5 expression and CD5 fluorescence ismaintained. The anti-CD5 binder is coupled to a second fluorophore,which has a different peak excitation wavelength than the firstfluorophore (attached to the anti-CD20 binder). The anti-CD5 antibody isadded to the sample, and then individual cells of the sample are exposedto a light source capable of exciting the second fluorophore, andfluorescent signal from individual cells is measured. Based on thefluorescent signal from cells, the average amount of CD5 in B-cells inthe sample is determined.

Although this example it described in the context of CD5, it should beunderstood that this concept of obtaining an approximate count to guidein addition of a desire amount of material for use in a subsequent step,is not limited to CD5 and use of this concept with other types of cells,analytes, or objects is not excluded.

Example 6

Blood cells may be imaged, identified, and quantified according to themethods disclosed herein. For example, two-dimensional images of cellsin a biological sample, where the cells are labeled (e.g., withfluorescent, chemiluminescent, enzymatic, or other labels) and plated(e.g., allowed to settle on a substrate) and imaged by a camera, may beprepared and analyzed as described in the present example. The cameramay include a lens, and may be attached to or used in conjunction with amicroscope. Cells may be identified in the two-dimensional images bytheir attached labels (e.g., from light emitted by the labels).

80 microliters of whole blood obtained from a fingerstick was loadedinto a capped vessel preloaded with 2 mg/ml EDTA. The capped vessel wascentrifuged at 1200×g for 5 minutes, to separate the blood cells fromthe blood plasma. Centrifugation of the capped vessel resulted in theseparation of the blood sample in the capped vessel into two majorcomponents (from top of the capped vessel to the bottom): 1) bloodplasma and 2) packed blood cells. This process ensures that no dropletsof blood remain isolated, but coalesce with the main body of the liquid.In addition, this process separates the cells from elements of theplasma thus reducing metabolism and allowing for longer storage of thesample.

The centrifuged capped vessel was loaded into a cartridge containingmultiple fluidically isolated reagents, tips, and a cytometry cuvette.The cartridge contained all the reagents required for the assay. Thecartridge was loaded into a device equipped with at least a centrifuge,a pipette and a platform to load the cuvette. The pipette in the devicehas a plurality of nozzles, some nozzles being of a different size thansome other nozzles.

Inside the device, a nozzle on the pipette was lowered on a cuvettecarrier tool causing it to engage a corresponding hole on the carriertool. This tool was subsequently moved to the cartridge and lowered onthe cytometer cuvette. Pins on the tool were then able to engagecorresponding holes on the cuvette and pick it up. The cuvette wastransferred to a loading station elsewhere in the device.

Next, inside the device, a larger nozzle of the pipette was lowered intothe cartridge to engage a pipette tip stored in the cartridge. Thepipette and tip together were then used to mix the cells and plasma inthe capped vessel by positioning the pipette tip within the sample inthe capped vessel and repeatedly aspirating material into and dispensingmaterial from the tip. Once the cells were resuspended in the plasma sothat the whole blood sample was thoroughly mixed, 5 microliters of themixed whole blood was aspirated to provide an aliquot for measurementsof properties of the blood sample. This 5 microliter aliquot was usedfor measurements directed to the red blood cells and platelets in thesample. As discussed below, a portion of the sample remaining afterremoval of this 5 microliter aliquot was used for measurements directedat white blood cells in the sample.

The 5 microliters of whole blood was dispensed into a vessel containinga mixture of phosphate buffered saline and 2% by weight of bovine serumalbumin, to dilute the whole blood twenty-fold (resulting in 100microliters of diluted sample). After mixing vigorously, 5 microlitersof this sample was transferred to another vessel containing a cocktailof labeling antibody reagents: anti-CD235a conjugated to alexa-fluor 647(AF647), anti-CD41 and anti-CD 61 conjugated to phycoerythrin (PE). Themixture was incubated for 5 minutes. Subsequently, 10 microliters ofthis mixture was mixed with 90 microliters of a buffer containing azwitterionic surfactant at <0.1% by weight. The surfactant moleculesmodify bending properties of the red cell membrane such that all cellsassume a stable spherical shape. This transformation is isovolumetric asthe buffer used is isotonic with cytoplasm and no exchange of fluid canoccur across the cell membrane. After incubating this for another 2minutes, 30 microliters of this solution was mixed with a solutioncontaining glutaraldehyde, a fixative and non-fluorescent beads of 10 umdiameter. The mixture had a final concentration of 0.1% glutaraldehydeand 1000 beads per microliter. Glutaraldehyde rapidly fixes cells thuspreventing cell lysis and other active biological processes.

In this non-limiting example, the pipette then engaged a tip in thecartridge, aspirated 7 microliters of the above mixture of and loadedthe 7 microliters into a channel within the cuvette placed on a platformwith the carrier tool. After the mixture was loaded in into cuvette, thepipette aspirated 10 microliters of mineral oil from a vessel in thecartridge, and placed a drop of mineral oil on both open ends of theloaded channel of the cuvette. Mineral oil was added to the ends of theopen channel to prevent evaporation of liquid from the loaded cuvettechannel. Next, the device-level sample handling apparatus engaged thecuvette carrier/cuvette combination, and transported the cuvettecarrier/cuvette combination from the module containing the cartridge tothe cytometry module of the device. At the cytometry module, thedevice-level sample handling apparatus placed the cuvettecarrier/cuvette combination on the microscopy stage of the cytometrymodule. The time required for these operations, in addition to a 2minute wait time allowed the swollen cells to settle to the floor of thecuvette prior to imaging.

After the cuvette carrier/cuvette was placed on the microscopy stage,the stage was moved to pre-determined location so that the opticalsystem of the cytometer could view one end of the channel containing thesample. At this location, the optical system relayed images of thesample acquired with darkfield illumination from a ringlight. Theseimages coupled with actuation of the optical system on an axisperpendicular to the plane of the cuvette were used to find the plane ofbest focus. Once focused, the optical system was used to acquirefluorescence images of the sample at different wavelengths, commensuratewith the fluorophores that were being used. For example, to visualizered blood cells that had been labeled with anti-CD235 conjugated toalexa fluor 647, a red (630 nm wavelength) light source was used toexcite the sample and wavelengths between 650 nm and 700 nm were used toimage the sample. A combination of a polychroic mirror and a bandpassemission filter was used to filter out unwanted wavelengths from theoptical signal. Since the cells had settled on the floor of the cuvette,images at a single plane of focus were sufficient to visualize all cellsin the region.

Data from the images was processed by a controller associated with thesample processing device. The image processing algorithms employed hereutilized fluorescence images of cells to detect them using a combinationof adaptive thresholding and edge detection. Based on local intensityand intensity gradients, regions of interest (RoI) were created aroundeach cell. Using darkfield images, beads in the sample were alsoidentified and RoIs were created around the beads. All the RoIs in eachfield of view were enumerated and their intensity in each image of thatfield of view were calculated. The information output by the imageprocessing algorithm consisted of shape or morphometric measurements andfluorescence and darkfield intensities for each RoI. This informationwas analyzed using statistical methods to classify each object as eithera red blood cell (positive for CD235a, but negative for CD41/CD61), aplatelet (positive for CD41/CD61 and negative CD235a) or a bead. Theshape descriptors such as perimeter, diameter and circularity were usedto calculate the volume of each red blood cell and platelet. Since thebeads were added at a known concentration, the average ratio of beads tocells over the whole channel was used to calculate cell concentration interms of cells/microliter. Based on the steps performed for processingthe sample, this concentration was corrected for dilution to arrive atconcentration of cells in the original whole blood sample. The followingquantities were calculated from a sample: 1) number of red blood cellsin the cuvette; 2) average volume of red blood cells in the cuvette; 3)red blood cell distribution width (RDW) of red blood cells in thecuvette; 4) number of platelets in the cuvette; and 5) average volume ofplatelets in the cuvette. Based on these calculations, the following wascalculated for the original blood sample.

Exemplary Measured Value Result Range Concentration of red blood cells(million cells per 4.8 4-6 microliter) Mean volume of red blood cells,femtoliter 88  80-100 red blood cell distribution width (RDW), (%) 12  11-14.6 Concentration of platelets (thousand cells per 254 150-400microliter) Mean volume of platelets, femtoliter 10.4  7.5-11.5

After removal of the 5 microliter aliquot used for analysis of RBC andplatelet information, the remaining 75 microliters of sample was used toanalyze the white blood cell population of the whole blood sample. Theremaining 75 microliters of whole blood had also been mixed byrepeatedly aspirating and dispensing the sample within the same thevessel by the pipette. Approximately 40 microliters of the remaining 75microliters of mixed whole blood was aspirated into a pipette tip, andtransferred by the pipette to a centrifuge tube in the cartridge. Thecentrifuge tube containing the blood sample was engaged by the pipette,and transferred to and deposited in a swinging bucket in a centrifugewithin the module. The centrifuge was spun to provide 1200×g for 3minutes, separating the blood into EDTA-containing plasma as thesupernatant and packed cells in the pellet.

After centrifugation, the centrifuge tube was removed from thecentrifuge and returned to the cartridge. The plasma supernatant wasremoved by the pipette and transferred to a separate reaction vessel inthe cartridge. From a reagent vessel in the cartridge, 16 microliters ofresuspension buffer was aspirated by the pipette, and added to the cellpellet in the centrifuge tube. The pipette then resuspended the cellpellet in the resuspension buffer by repeatedly aspirating anddispensing the mixture in the centrifuge tube. Next, the pipetteaspirated 21 microliters of the resuspended whole blood and added it toanother vessel containing 2 microliters of anti CD14-pacific blue anddraq5, mixed, and incubated for 2 minutes. Twenty microliters of thismixture was then added to 80 microliters of a lysis buffer. The lysisbuffer is a solution of a gentle surfactant such a saponin inconjunction with a fixative such as paraformaldehyde. The detergentcauses a large number of holes to be formed in the membranes of cells.Red blood cells, due to their unique membrane properties, areparticularly susceptible to this hole formation and lyse completely,their contents leaking out into the liquid around. Presence of thefixative prevents unintentional lysis of the white blood cells.Platelets also remain unlysed. The purpose of this step is to remove redblood cells from the mixture as they outnumber white blood cells byabout 1000:1. Platelets do not interfere with imaging and hence areirrelevant to this process. The lysis buffer also contained 10 μMnon-fluorescent beads at a known concentration.

After a 5 minute incubation, the vessel was spun again at 1200×g for 3minutes. The supernatant was aspirated by a pipette tip, removing thered blood cell ghosts and other debris, and deposited into a waste areain the cartridge. Approximately 15 microliters of liquid with packedwhite blood cells were present in the cell pellet.

In order to determine a rough approximation of the number of white bloodcells present in the cell pellet, the pipette first resuspended thewhite blood cells in the vessel and then aspirated the liquid,transferred it to spectrophotometer in the blade The white blood cellsuspension was illuminated with light at a wavelength of 632 nm, whichis the excitation wavelength for alexa fluor 647 dye and draq5. Thelight emitted by the cell suspension was filtered by a 650 nm long passfilter and measured in the spectrophotometer. This measurement wascorrelated with previously generated calibration curve to estimate arough concentration of white blood cells in the cell suspension.Typically, cell concentrations ranged from about 1000 cells permicroliter to about 100,000 cells per microliter. This estimate was usedto calculate an appropriate dilution factor to ensure that theconcentration of cells in the cuvette was constrained to within atwo-fold range around a pre-defined target concentration. The purpose ofthis step was to ensure that cells are not present at too high or toolow a density on the cuvette. If the cell density is too high, theaccuracy of image processing algorithms is compromised, and if the celldensity is too low, an insufficient number of cells are sampled.

Based on the dilution factor calculated in the above step, a diluentcontaining labeled antibodies against CD45 (pan-leukocyte marker), CD16(neutrophil marker) and CD123 (basophil marker) was added to the cellsuspension and mixed.

Once the cuvette in complex with cuvette carrier was placed on thecuvette carrier block, 10 microliters of the mixture of white bloodcells resuspended in cytometry buffer was loaded into each of twochannels in the cuvette. After the mixture was loaded into channels ofthe cuvette, the pipette aspirated 10 μl of mineral oil from a vessel inthe cartridge, and placed a drop of mineral oil on both open ends ofboth channels in the cuvette loaded with white blood cells.

Next, the device-level sample handling apparatus engaged the cuvettecarrier/cuvette combination, and transported the cuvette carrier/cuvettecombination from the module containing the cartridge to the cytometrymodule of the device. At the cytometry module, the device-level samplehandling apparatus placed the cuvette carrier/cuvette combination on themicroscopy stage of the cytometry module. After the cuvettecarrier/cuvette was placed on the microscopy stage, the two channels ofthe cuvette containing white blood cells were imaged as described abovefor the RBC/platelet mixture.

Darkfield images of the white blood cells were used to count the numbersof cells in a field (as shown in FIG. 9A). Cell surface markers wereused to determine the cell type of individual white blood cells in animage; for example, CD14 marks monocytes; CD123 marks basophils; CD16marks neutrophils; and CD45-AF647 were used to mark all leukocytes(FIGS. 9B-9E). The nuclear stain Draq5 was used to mark cell nuclei, andso to differentiate nucleate cells (such as white blood cells) frommature red blood cells, which have no nucleus (FIG. 9F).

The image processing algorithms employed here utilized fluorescenceimages of cells to detect them using a combination of adaptivethresholding and edge detection. Based on local intensity and intensitygradients, boundaries of regions of interest (RoI) were created aroundeach cell. Using darkfield images, beads in the sample were alsoidentified and RoI boundaries were created around the beads. All theRoIs in each field of view were enumerated and their intensity in eachimage of that field of view were calculated. The information output bythe image processing algorithm consisted of shape or morphometricmeasurements and fluorescence and darkfield intensities for each RoI.This information was analyzed using statistical methods to classify eachobject as a lymphocyte, monocyte, basophil, eosinophil, neutrophil or abead. Based on enumeration of cells of different types, thecorresponding bead count and the dilution ratio implemented duringsample processing, an absolute concentration of cells per microliter oforiginal whole blood was calculated. This was calculated for all whiteblood cells and each subtype, and reported as both absoluteconcentration (cells per microliter) and proportion (%).

Examples of images and plots of results of such measurements arepresented in FIGS. 9, 10, and 11.

FIG. 9 shows representative images of blood cells from a sample of wholeblood; these images were taken using different imaging techniques anddyes. The image shown in FIG. 9A was taken of cells from whole bloodusing dark-field illumination. The image shown in FIG. 9B was taken ofcells from whole blood showing fluorescence from anti-CD14 antibodieslabeled with PAC Blue dye; the fluorescent cells are monocytes. Theimage shown in FIG. 9C was taken of cells from whole blood showingfluorescence from anti-CD123 antibodies labeled with PECy5 dye; thefluorescent cells are basophils. The image shown in FIG. 9D was taken ofcells from whole blood showing fluorescence from anti-CD16 antibodieslabeled with PE dye; the fluorescent cells are neutrophils. The imageshown in FIG. 9E was taken of cells from whole blood showingfluorescence from anti-CD45 antibodies labeled with AF647 dye; allleukocytes fluoresce under these conditions. The image shown in FIG. 9Fwas taken of cells from whole blood dyed with Draq5 to stain cellnuclei. Thus, leukocytes and platelets are stained and fluoresce underthese conditions, but red blood cells (lacking nuclei) are not stainedand do not fluoresce.

FIG. 10 shows a representative composite image of cell-types in wholeblood from images acquired according to the methods disclosed herein.Images of a monocyte (labeled and seen in the upper left quadrant of thefigure, with a reddish center surrounded by a blue-purple ring), alymphocyte (labeled and seen in the center of the figure, with a brightred center surrounded by a dimmer red ring), an eosinophil (labeled andseen in the lower left quadrant of the figure, with a green centersurrounded by a red border), and a neutrophil (labeled and seen in thelower right quadrant of the figure, with a green center surrounded by ayellow and green border) are shown in the figure.

It is of interest to identify and quantify various cell types found insuch blood samples. There may be multiple ways to approach such aclassification process, which, in some embodiments, may be considered asbeing a statistical problem for multi-dimensional classification. Itwill be understood that a wide variety of methods are available in thefield to solve these types of classification problems. A particularembodiment of such an analysis is provided below.

FIG. 11 shows plots of various cell types identified and quantified bythe cytometric assays described in this example. FIG. 11A shows a plotof spots (cells) by intensity of the marker FL-17 (anti-CD14 antibodylabeled with pacific blue dye) versus intensity of FL-9 (darkfieldscatter signal) to identify monocytes. FIG. 11B shows a plot of spots(cells) by intensity of the marker FL-19 (anti-CD123 antibody labeledwith PE-CY5 dye) versus intensity of the marker FL-15 (anti-CD16 labeledwith PE dye) to identify basophils. FIG. 11C shows a plot of spots(cells) by intensity of the marker FL-15 (anti-CD16 labeled with PE dye)versus intensity of the marker FL-11 (anti-CD45 antibody labeled withAF647 dye) to identify lymphocytes. FIG. 11D shows a plot of spots(cells) by intensity of the marker FL-15 (anti-CD16 labeled with PE dye)versus intensity of FL-9 (darkfield scatter signal) to identifyneutrophils and eosinophils.

The initial identification of monocytes (9.6%, as shown in FIG. 11A) isused to guide the subsequent identification of basophils (0.68%, asshown in FIG. 11B). The identification of monocytes and basophils asshown in FIGS. 11A and 11B is used to guide the subsequentidentification of neutrophils and eosinophils (68% neutrophils, 3.2%eosinophils, of the WBCs shown in FIG. 11D). Finally, lymphocytes areidentified as shown in FIG. 11C (93% of the WBCs plotted in FIG. 11C,corresponding to 18% of the cells in the original sample).

The present methods correlate well with other methods. Counts of whiteblood cells, red blood cells, and platelets were made with samples ofEDTA-anti coagulated whole blood. The white blood cells were furthercounted to determine the numbers of neutrophils, monocytes, andlymphocytes in the sample. In the measurements shown in FIG. 12,EDTA-anti coagulated whole blood samples were split into two, and onepart of the samples were run on the system disclosed herein, using themethods disclosed herein. The other part of the samples was run on anAbbott CELL-DYN Ruby System (Abbott Diagnostics, Lake Forest, Ill.,USA), a commercial multi-parameter automated hematology analyzer. Acomparison of the results obtained with both methods is shown in FIG.12.

As shown in FIGS. 12A-12C, the numbers of white blood cells (“WBCs”,FIG. 12A), red blood cells (“RBCs”, FIG. 12B) and platelets (FIG. 12C)measured by the present methods correlate well with the numbers of WBCs,RBCs, and platelets measured by other methods in corresponding aliquotsof the same samples as were analyzed by the present methods. As shown inFIGS. 12D-12F, the numbers of neutrophils, monocytes, and lymphocytesmeasured by either method were very similar, and correlated well witheach other.

In aspects of the term as used herein, the term “cytometry” refers toobservations, analysis, methods, and results regarding cells of abiological sample, where the cells are substantially at rest in a fluidor on a substrate. Cells detected and analysed by cytometry may bedetected and measured by any optical, electrical or acoustic detector.Cytometry may include preparing and analyzing images of cells in or froma biological sample (e.g., two-dimensional images). The cells may belabeled (e.g., with fluorescent, chemiluminescent, enzymatic, or otherlabels) and plated (e.g., allowed to settle on a substrate) and,typically, imaged by a camera. A microscope may be used for cell imagingin cytometry; for example, cells may be imaged by a camera and amicroscope, e.g., by a camera forming an image using a microscope. Animage formed by, and used for, cytometry typically includes more thanone cell.

Optical Systems

Referring now to FIG. 6, one embodiment of an optical system suitablefor use herein will now be described. Although this embodiment of thesystem is described in the context of being able to perform cytometry,it should also be understood that at least embodiments of the systemalso has capability beyond cytometry. By way of example and notlimitation, the system can have application outside of cytometry due tothe imaging and image processing capabilities associated with someembodiments. Since images are captured of the sample being analyzed andimage information is typically linked or associated in the system toquantitative measurements, one can further analyze the images associatedwith the quantitative information to gather clinical information in theimages that would otherwise be unreported.

The embodiment shown in FIG. 6 shows a perspective view of a cuvette 600that has a plurality of openings 602 for receiving sample for analysis.Although the system is described in the context of a cuvette, it shouldbe understood that other sample holding devices may also be used inplace of or in combination with the cuvette 600.

As seen in the embodiment of FIG. 6, the openings 602 may allow for asample handling system (not shown) or other deliver system to depositsample into the opening 602 which may then lead to an analysis area inthe cuvette where the sample can be analyzed. In one nonlimitingexample, the analysis area may be a chamber. In another nonlimitingexample, the analysis area may be a channel. In a still furthernonlimiting example, the analysis area may be a channel wherein thesample is held in a non-flowing manner. In any of the embodimentsherein, the system can hold the samples in a non-flowing manner duringanalysis. Optionally, some alternative embodiments may be configured toenable sample flow through the analysis area before, during, or afteranalysis. In some embodiments, after analysis, the sample is extractedfrom the cuvette 600 and then delivered to another station for furtherprocessing and/or analysis. Some embodiments may use gate(s) in thesystem to control sample flow.

FIG. 6 shows that some embodiments of cuvette 600 have a plurality ofopenings 602. Embodiments having more or fewer openings 602 in thecuvette 600 are not excluded. Some embodiments may link certain openings602 such that select pairs or other sets of openings 602 can access thesame channel. By way of nonlimiting example, there may an opening 602 ateach end of an analysis area. Optionally, more than one opening 602 maybe at one end of the analysis area.

Some embodiments may provide structures 604 over select areas of thecuvette 600. In one embodiment, the structures 604 are ribs that providestructural support for areas of the cuvette that are selected to have adefined thickness. The structures 604 may be use when the definedthickness areas are at a reduced thickness relative to certain areas ofthe cuvette and thus could benefit from mechanical support provided bystructures 604.

In some embodiments, these controlled thickness areas are selected to bepositioned over the analysis areas. In some embodiments, thesecontrolled thickness areas can impart certain optical properties over ornear the analysis areas. Some embodiments may configure the structures604 to also impart optical properties on light passing through thecuvette 600. Optionally, some embodiments may configure the structures604 to not have an impact on the optical qualities of the cuvette 600.In such an embodiment, the structures 604 may be configured to have oneor more optically absorbent surfaces. For example and not limitation,certain surfaces may be black. Optionally, some embodiments may have thestructures 604 formed from a material to absorb light. Optionally, thestructures 604 can be positioned to provide mechanical support but donot interact with the optical properties of cuvette 600 near theanalysis areas.

Some embodiments of cuvette 600 can be configured to have structures 610that allow for a sample handling system to transport the cuvette 600. Inone nonlimiting example, the structures 610 can be openings in thecuvette 600 that allow for a pipette or other elongate member to engagethe cuvette 600 and transport it to the desired location. Optionally, inplace of or in combination with said opening(s), the structures 610 canbe a protrusion, hook, and/or other non-negative feature that can beused to engage a cuvette transport device.

It should be understood that the cuvette 600 is typically formed from anoptically transparent or transmissive material. Optionally, only selectportions of the cuvette 600 such as the analysis areas or areasassociated with the analysis areas are optically transparent.Optionally, select layers or areas in the cuvette 600 can also beconfigured to be non-light transmissive.

FIG. 6 shows that in this embodiment, the cuvette 600 rests on a supportstructure 620 wherein at some or all of the support structure 620 isformed from an optically transparent or transmissive material. In someembodiments, the optically transparent or transmissive portions areconfigured to be aligned with the analysis areas of the cuvette 600 toallow for optical interrogation of the sample in the analysis area. Inone nonlimiting example, the support structure 620 can be movable in theX, Y, and/or Z axis to move the cuvette 600 to a desired position forimaging. In one some embodiments, the support structure 620 comprises aplatform or stage that moves only in two of the axes. Optionally, somesupport structures may move only in a single axis. The cuvette 600 canbe configured to be operably coupled to the support structure 600through friction, mechanical coupling, or by retaining members mountedto one or both of the components.

FIG. 6 further shows that for illumination for darkfield and/orbrightfield observation, there may be an illumination source 650 such asbut not limited to a ringlight below the support structure 620 to locateillumination equipment below the level of the cuvette 600. This leavesthe upper areas of the cuvette 600 available for pipettes, samplehandling equipment, or other equipment to have un-hindered access toopenings or other features on a top surface of the cuvette 600.Optionally, some embodiment may locate an illumination source 660 (shownin phantom) above the cuvette 600 to be used in place of, in single, orin multiple combination with underside illumination source 650. Anobjective 670 can be positioned to observe the sample being illuminated.It should be understood that relative motion between the cuvette 600 andthe optical portions 650 and 670 can be used to allow the system tovisualize different analysis areas in the cuvette 600. Optionally, onlyone of components is in motion to interrogate different areas of thecuvette 600.

Referring now to FIG. 7, one embodiment of a suitable imaging systemwill now be described in more detail. FIG. 7 shows a schematiccross-sectional view of various components positioned below the supportstructure 620. The cross-section is along the area indicated by bentarrows 7 in FIG. 6.

FIG. 6 shows that in the present embodiment, the cuvette 600 comprises abase portion 606 and analysis areas 608 defined by a cover portion 612.Optionally, the analysis areas 608 may be defined within a single piece.Optionally, the analysis areas 608 may be defined by using more than twopieces, such as but not limited a discrete cover piece for each of theanalysis areas 608. In one embodiment, the layer 606 comprises opticallyclear plastic such as but not limited to cyclo olefin polymerthermoplastic which deliver superior optical components andapplications. Some may form one or more layers or components from glass,acrylic, clear polymer, or other transparent material.

In this nonlimiting example, the sample to be interrogated can be housedin whole or in part in the area 608. By way of non-limiting example, theoptics below the support structure 620 may include a ringlight 650 thatcomprises a toroidal reflector 652 and a light source 654. Otherillumination components suitable for darkfield illumination are notexcluded. Some embodiments may use a mirror. Some embodiments use acoated reflective surface. Some embodiments may use a differentreflector and not a toroidal reflection. Some embodiments may use aparabolic reflector. Some embodiments may use a parabolic reflector inthe shape of an elliptic paraboloid. Some embodiments may use aplurality of individual reflector pieces. Some embodiments may not useany reflector. Some embodiments obtain oblique illumination through theuse of angled light sources positioned to direct light with or withoutfurther assistance from one or more external reflectors.

The embodiment of FIG. 6 shows excitation energy sources 680, 682, and684 such as but not limited laser diodes at specific wavelengths thatare mounted to direct light into the sample in analysis area 608. In onenonlimiting example to facilitate compact packaging, the energy sources680, 682, and 684 may direct light to a dichroic 690 that then directsthe excitation wavelengths into the analysis area 608. The excitationwavelength(s) cause fluorescence wavelengths to be emitted byfluorophores in marker(s), dye(s), and/or other materials in the sample.The emitted fluorescence wavelengths are funneled through the objective670, through the dichroic 690, through an optional filter wheel 692, andinto a detector 700 such as but not limited to a camera system. By wayof nonlimiting example, the dichroic 690 is configured to reflectexcitation wavelengths but pass fluorescence wavelengths and anywavelengths desired for optical observation.

In one embodiment, all fluorescence excitation wavelengths areilluminating the sample in analysis area 608 simultaneously. Thedetector 700 may be coupled to a programmable processor 710 that cantake the captured signal and/or image and deconstruct which wavelengthsare associated with which fluorophores that are fluorescencing. Someembodiments may have the excitation sources illuminate the samplesequentially or in subsets of the entire number of excitation sources.Of course, it should be understood that the system is not limited tofluorescence based excitation and that other detection techniques andexcitation techniques may be used in place of or in single or multiplecombination with fluorescence. For example, some embodiments may alsocollect darkfield illumination scatter information simultaneously orsequentially in combination with fluorescence detection.

Referring now to FIG. 8A, a still further embodiment will now bedescribed. FIG. 8A shows a schematic of a cross-section of a portion ofthe cuvette 600 and the dark field scatter illumination source such asbut not limited to the ringlight 650. For ease of illustration, thesupport structure 620 is not shown. As seen in FIG. 8A, the ringlight650 provides illumination for the analysis area 608. In the presentembodiment, the ringlight components 652 and 654 are shown. The lightsource 654 may be white light or light sources such as but not limitedto LEDs or laser diodes with specific wavelength output or outputranges. Optionally, the ring of light source 654 could be fiber opticcable with many splice to create a ring of light. Optionally, the lightsource 654 may be an LED which has specific narrow divergence anglecontrolled by the reflector. It may be desirable to control divergenceangle from the ringlight through the selection of light source and/ordesign of the reflector.

By way of nonlimiting example, laser illumination as the source 654provides for narrow light pattern with results in lower transillumination in the present epi-style lighting configuration (whereillumination components are all on one side of the sample) but becausethe source is a coherent source, it also lowers background signallevels. Laser illumination may not have adjacent channel illuminationthat typically occurs with more diffuse light sources and thus less,laser illumination can result in less trans illumination. Of course, itis desirable that the decrease in trans illumination is less than thedecrease in background, where the more significant drop in backgroundresults in a more distinguishable signal. Optionally, LED as theillumination source 654 provides for a diffuse light pattern, withincreased background and increased trans illumination. Of course, it isdesirable that the increase in trans illumination is greater than theincrease in background.

Some cuvette embodiments may include cuvettes formed from a plurality ofindividual layers adhered together, having the cuvette molded from oneor more materials, and/or having reflective layers added to the cuvetteat different surfaces to enhance multiple TIR.

Because the present embodiment may be operating in combination withfluorescence, desirable that our darkfield illumination is not whitelight. Some alternative embodiments may use just white light if theirsystem is not using fluorescence detection in combination with darkfieldand/or brightfield microscopy.

FIG. 8A shows that in some embodiments, the device may have layers inthe cuvette 600 that are optically non-transmissive such as layer 800.This may be useful in embodiments where the light source 654 is diffuseand light is not directed to specific locations. The layer 800 can blocklight that are not entering the cuvette 600 at desired angles and/orlocations. The layer 800 can be configured to be positioned to preventillumination except through the area below the analysis areas 608. Somemay only have specific areas that are blacked out nearest the analysisareas 608. Some embodiments may have blacked out or non-tranmissivematerial in more than one layer. Some may have blacked out ornon-tranmissive material in different orientations, such as but notlimited to one being horizontal and one being vertical ornon-horizontal.

FIG. 8A shows that total-internal-reflection (TIR) may be present at anupper surface 614 and/or at surface 618 in one or more of the supportstructures 604. TIR is a tunable feature that can selected based on thematerial used for the cuvette 600 and the geometry and/or thickness ofthe controlled thickness area 613 of the cuvette 600. The presence ofTIR which allows for oblique angle illumination coming from above thesample is desirable, particularly for darkfield microscopy. In someembodiments, it is desirable to maximize TIR from above the sample.Optionally, some embodiments may only have TIR from surfaces over theanalysis areas 608. Optionally, some embodiments may only have TIR fromsurfaces over the controlled thickness areas 613. Optionally, someembodiments not have TIR from the support structures 604. Optionally,some embodiments not have TIR from surface 618. Optionally, someembodiments may have TIR from other surfaces in the cuvette 600, so longas it is scatter light as oblique angles being directed back to theanalysis area 608.

Optionally, some embodiments may put reflective material at surfaces 614and/or 618. Optionally, only surface 614 has reflective material on thesurface. Optionally, surface 618 may be treated to be black so as to belight absorbing. Some embodiments may select the width of the controlledthickness area 612 to be wider than the analysis area 608. For someembodiments using laser illumination, the layer 800 may be removed or belight transmitting as the laser illumination is sufficiently focused soas not to require blackout between analysis areas 608.

By way of example and not limitation, the use of TIR can also enablelight 820 from adjacent areas to be directed into the analysis area 608.Under traditional terminology, this is trans illumination. Line 830shows light coming directly from the ringlight and not by way of TIR,and this is epi illumination. The combination of both types of lightcomponents from a light source located below the sample (or only oneside of the sample) allows for improved performance as compared tosources that can only provide one of those lighting components. This isparticularly useful for darkfield microscopy.

One nonlimiting example of the use of the embodiment shown in FIG. 8 isdarkfield illumination to measure scatter properties of cells in thesample. Darkfield microscopy is an age old method that has been usedmainly as a contrast enhancing technique. Since only the light scatteror reflected by the sample is imaged, the image background is fullydark. Quantitative darkfield microscopy has not been used to measurescatter properties of cells comparable to the traditional “side scatter”parameter in flow cytometers.

From the hardware perspective, illumination for darkfleld microscopy isdesired to be oblique, i.e. no rays of light from the illumination lightsource should be able to enter the objective without contacting thesample first. By way of example and not limitation, illumination shouldbe at a wavelength that does not excite any other fluorophores alreadypresent in the sample. Optionally, this illumination allows for the useof high numerical aperture (NA) lenses for imaging. By way of exampleand not limitation, for traditional lens sizes associated with opticalmicroscopes, the NA may be at least 0.3. Optionally, the NA is at least0.4. Optionally, the NA is at least 0.5. Optionally, some embodimentsmay use oil immersion objective lenses to obtain a desired NA,particularly when lens size is limited below a certain level.

Traditional methods for darkfield illumination have usedtrans-illumination, where the sample is between the imaging lens anddarkfield light source. Thus, in this embodiment, the detection andillumination components are not on the same side of the sample. Theepi-illumination methods (where the imaging lens/objective and thedarkfield light source are on the same side of the sample) require theuse of specially manufactured objectives and typically do not allow theuse of high NA objectives, thus limiting the capabilities of the wholesystem.

By contrast, at least some embodiments of darkfield illumination systemsdescribed herein have the following attributes. In terms of hardware,the scheme of this embodiment of FIG. 8A is “epi” in that the ringlightused for darkfield illumination is on the same side of the sample as theobjective. This can be desirable from the system-perspective, althoughalternative embodiments with light sources on the other side may be usedalone or in combination with the embodiments described herein. In onenonlimiting example, the ringlight is designed such that the LEDs and/orlasers of the light source 654 are all in the same plane and have thesame orientation (horizontal plane and directing light upwards). Someembodiments may have light in the sample plane but directing light in anon-parallel manner, such as but not limited to a cone-like manner. Someembodiments may have light in different planes but directing light inthe same orientation. Some embodiments may have light in differentplanes but directing light in a non-parallel manner, such as but notlimited to a cone-like manner. The light is reflected by a toroidalmirror 652 to achieve oblique illumination of the sample.

In addition to the ringlight and the toroidal reflector, the opticalproperties of the cuvette 600 shown in the embodiment of FIG. 8 alsosignificantly affects darkfield illumination. In this embodiment, thecytometry cuvette 600 is designed such that light coming from theringlight 650 is allowed to fall directly on the sample; but in additionto this, light is also “reflected” on the sample from features of thecuvette so as to emulate “trans” illumination. This reflection can be byway of TIR and/or true reflection.

Note that any trans-illumination scheme allows one to measure forwardscattered light from a particle whereas an epi-scheme allows one tomeasure only the back-scattered light. Forward scattered light isgenerally two orders of magnitude greater in intensity than theback-scattered light. Trans-scheme thus allows the use of much lowerillumination intensities and reduces harmful side effects on the sample.

As seen in the embodiment of FIG. 8A, the ringlight 650 and cuvette 600provide a system that can be tuned such that the intensities of transand epi illumination are adjusted for improved performance overtraditional epi illumination. This tuning can be achieved by virtue ofcuvette geometry to control angles and extent of total internalreflection and material properties.

FIGS. 8B and 8C show that the sample holder such as cuvette 600 istransported from one location such as where sample preparation may occurand then to the detector D as seen in FIGS. 8B and 8C. The cuvette 600does not release fluids into or onto the detector D, but instead isself-contained unit that keeps all of the sample therein. There may beone or more, two or more, or three or more locations on the detector Don which there is transparent surface on which the cuvette 600 or othersample holder can engage to provide a transparent interface for samplesignal detection to occur. The elements of FIG. 8C can be found inreference to U.S. patent application Ser. No. 13/769,779 fullyincorporated herein by reference.

Darkfield

At least some embodiments herein include a dark field illuminationsource and cuvette. The relevant features of the cuvette 600 relate todesigning the cuvette dimensions and optical materials and the geometryof the cuvette. The cuvette increases the extent of darkfieldillumination through total internal reflection (TIR) and/or purereflection. In one embodiment, the system may simultaneously use transdarkfield and epi darkfield.

In some embodiments herein, the cuvette combined with the light sourceenables trans and epi illumination using only physical system in epiconfiguration (light source on one side of sample). The basic cuvette isdesigned to contain the biological sample and present it forvisualization. In one embodiment, the coverslip 612 may have a specificdesign. Materials have different index of refraction. Some embodimentsmay make cover slip 612 of glass.

One can design the material of the top coverslip 612 to facilitateillumination and image collection. To get light to the cells, theringlight 650 may be circular, have light sources 654 position in adiscrete or continuous pattern, and use a curved reflector 652 to directlight to the sample.

In darkfield microscopy, the sample is illuminated by oblique rays. Thelight going into the microscopy is the light scattered by the sample.Measuring scatter properties of the cells. If nothing is there, theimage is black.

In the present non-limiting example, the reflector 652 and LED 654 ofthe ringlight 650 are designed to reflect so that a minimum fractiongoes directly back into the objective as non-specific background. Thesystem is designed to give TIR surface and reflection from othersurfaces back into the target area 608. The cells in the sample in 608is getting light directly from the ringlight from underneath the cell(this is epi). There is also light coming from the top surfaces(reflected) and this is trans.

With the ringlight 650 in the same position, one now has light comingfrom two directions from a single ringlight source. This is all oblique.One can control the relative strengths of the two light components bydesign of the cuvette and material used for the cuvette.

This darkfield illumination is different from conventional darkfield. Byway of nonlimiting example, this embodiment may use a reflective layeron the backside of certain surfaces of the coverslip 612 to reflect allof the light. Some embodiments may use a full or selectively reflectivebackground.

In the present embodiment, the light is desirable at an oblique anglewhich keeps illumination darkfield. Some may angle the light sources 654at an angle and thus not use the reflector 652. The reflector 652 mayimprove manufacturability of the light source 654 since all lights arein the same plane, directed in the same direction. Optionally, theangled light sources 654 may also be used in place of or in combinationwith a reflector.

It should be understood that here even though trans component may be inone example 10 times weaker than epi illumination component, the scatterfrom the cells in the sample due to trans may be 200 times stronger fromthe same amount of epi versus scatter from the same amount of trans. Andthus, the small amount of trans can significantly enhance the scatterfrom cells. The light collected from epi illumination also does notinclude defraction. Defraction is a substantial component of scatter andthe use of trans illumination provides for some amount diffraction.Thus, there is reflective, refractive, and defractive components whenusing trans and epi illumination. With epi alone, there may be onlyreflective. Traditional methods uses all trans darkfield illuminationwhich takes significant amount of space to configure, due to componentsbeing on both sides of the sample. The present embodiment may obtain thespace savings of an epi configuration but still have epi and transillumination components on the sample.

Designing the sample holder and the light source together can enable anepi configuration to increase the amount of trans illumination,particularly uniform trans illumination. Some embodiment may usemirrored surfaces but TIR can be tuned to create the desired translighting that is uniform and at oblique angles into the analysis areafor darkfield illumination of the sample. In one nonlimiting example, athicker top 612 allows the TIR to come back into the target area 608.Traditional hardware may have some TIR but the light may not come backinto the area 608. Additionally, not just that TIR illumination comesback into the channel but that it comes back uniformly. This embodimentof FIG. 8 has certain surfaces at certain angles, has certain blacksurface(s), and certain reflective surface(s) so that the light comesback uniformly. Optionally, one could put a fully reflective surface ona top (such as but not limited to a flat top but optionally over selectareas of top 612 such as area 613).

By way of nonlimiting example, embodiments here take an imaging basedplatform and instead of using a high complication, high cost systemwhich may for example have 16 laser, the present embodiment leverages amore integrated detection system to be able to pick-up the differentialsof cells and types.

In one nonlimiting example, it is the combination of all these differenttypes of information to achieve the clinical goals. This may includequantitative and/or qualitative linked to quantitative, or images linkedto quantitative measurements. Not only different channels offluorescence where each channel may have one or more specific molecularmarkers targeted and that is quantitative information, but withmicroscopy, some embodiments herein have the ability to look at thebackground that staining forms inside the cell (whether it is in thecytoplasm, is it concentrated on the surface, in the nucleus,) that canlink image and/or qualitative information that generated thequantitative measurements. In this manner, the linkage of the originalimages that created the quantitative results are available for furtheranalysis if it turns out that the quantitative measurements triggeralarms or meet thresholds the suggest further analysis is desired.Embodiments herein can interrogate background staining creates in thecell. One can image if the staining is in the cell, the cytoplasm, etc.. . .

Some embodiments herein may be combining the quantitative scatterproperties of the cell, the shape of the cell, and/or the size of thecell. Some embodiments here measure the physical properties, opticalproperties, and bio/biochemical properties all in the same device at thesame time. All can be combined in a programmable processor or otherprocessing system link the various types of information to achieve theclinical goal of the assays.

Many traditional devices do one or the other. They do not do both andthere is also no linkage between different types of information. Someembodiments herein, where image information is retrievable thatgenerated the quantitative measurements, can be extended to tissuemorphology measurement. Optionally, the system can be applied to papsmear, which is more similar to traditional cytology. It can be extendedto anything done using traditional microscopy. In urine, at least someof the present embodiments can look at and analyze crystals and not justcells. One can look at crystals of inorganic salts and chemicals fromurine samples that had created certain quantitative readings on oneportion of a graph, such as but not limited what may be seen in FIG. 1Awhere different regions of data are circled. Image information forcertain data regions can be retrieved to further analyze the underlyingcell images that created the measurements plotted on the graph or chart.

Some embodiments herein combine the imaging features with the pathologyfeatures. For example, tissue prep may occur inside a blade or module,and such prepped material can be imaged in this platform. Then theimages or analysis is sent to servers to do image analysis to dodiagnosis or digital pathology to enable a pathologist to do analysis.

Esoteric Cytometry and Specialty Cytometry Marker

Many traditional advanced or esoteric cytometric assays require atraditional system to measure a large number of markers on cells,typically simultaneously. The general approach in the field have beentied to high capability instruments such as six or other multiplenumbers of lasers and 18 different PMT tubes to measure all of theseparameters simultaneously. Part of it has been dictated by traditionalmethodology of identifying all markers on a cell at the same time, whichhas driven it. However, in many clinical settings, this simultaneousmeasurement is not the requirement. In many clinical requirements, oneis interested in seeing how many cells are positive for one marker, howmany are positive for a combination of two or three markers. Someembodiments herein provide for multiple combinations of staining schemeswhere one may have a set of, for example, 10 markers, where one cancombine them in sets of 3-4 or 5-6 markers where one can combine themsuch that even if combining two markers in the same color, someembodiments of the present system can de-convolute which signal camefrom which marker. This allows some embodiments of the present system toreduce the hardware requirements in terms of the number of lightsources, the number of channels used for sample analysis. Thus, usingsubsets or markers in non-simultaneous manner in a pre-determinedpairing can be useful to enable esoteric cytometry. Perhaps certainmarkers are “gating” markers and they can be tested first and if theresults are negative, then other follow-on markers may not be need. Someembodiments herein using this non-simultaneous system also reduces thesample volume requirement.

It should be understood that by using imaging, the ability to get anactual count, it may be more accurate than traditional cytometry.Traditional flow cytometry gating does not allow for actual count.Imaging can actually be more accurate. The gating in flow cytometry issubjective and thus this can vary from system to system.

Some embodiments herein may also gate, but the gating is basedalgorithmically based on various factors including but not limited topatient health. Classification means is trained on a population ofpatients knowing if they are healthy or diseased. Some embodiments herecan flag a patient that is abnormal and flagging it for review. Selflearning gating can determine if different gating is desired based oninformation conveyed regarding the patient health. Thus, the gating forsome embodiments herein for the sample is done algorithmically, possiblywith a programmable processor, and the gating changes based on patienthealth.

Imaging: in many cases, one may want to minimize hardware capability andto re-use the sample volume. Thus, the more capability one can extractfrom the imaging, the better in terms maximizing information from evenless sample. Thus, the more information one can get to differentiatedifferent cell types from minimum number of pictures, the more one mayminimize the sample volume required.

Optionally, in one non-limiting example, the cuvette for use in themicroscopy stage can be configured as follows. The middle channel layercomprises a core of thin plastic membrane 800 withpressure-sensitive-adhesive (psa) on both sides. One side adheres to thewindow-layer 606 and the other side to the molded-top-layer. The core isan extruded film that is black in color, primarily due to opticalreasons of preventing light scatter and optical cross-talk between thedifferent liquid channels. This core membrane has to have good (small)thickness control and is typically formed from an extruded film of blackPET or black HDPE (polyethylene). The psa sub-layers on both sides haveto be as thin as possible for preserving the tight thickness control ofthe overall liquid channel, yet thick enough to provide a good fluidicseal around the liquid channel. The psa adhesives found best for ourdesign were acrylic in nature and had high adhesion strength forlow-surface-energy plastics. The liquid channels, ports and otheralignment features on the middle layer were fabricated usinglaser-cutting or die-cutting processes.

This embodiment also shows that magnetic feature(s) such as but notlimited to pucks or discs may be incorporated into the cuvette, such asbut not limited to being the molded top layer. This can be used tosimplify hardware use to transport the cuvette. Here, the handlingsystem can engage the magnetic features in the cuvette to transport itwithout having to add an additional sample handling device.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, different materials may be used to create differentreflective surfaces in the cuvette or other surfaces along a lightpathway in the optical system. Optionally, the reflective surface isselected so that the reflection is only diffusive. Optionally, thereflective surface is selected so that the reflection is only specular.Some embodiment may use a flat top illumination scheme as set forth inCoumans, F. A. W., van der Pol, E., & Terstappen, L. W. M. M. (2012),Flat-top illumination profile in an epifluorescence microscope by dualmicrolens arrays. Cytometry, 81A: 324-331. doi: 10.1002/cyto.a.22029,fully incorporated herein by reference for all purposes.

Additionally, concentrations, amounts, and other numerical data may bepresented herein in a range format. It is to be understood that suchrange format is used merely for convenience and brevity and should beinterpreted flexibly to include not only the numerical values explicitlyrecited as the limits of the range, but also to include all theindividual numerical values or sub-ranges encompassed within that rangeas if each numerical value and sub-range is explicitly recited. Forexample, a size range of about 1 nm to about 200 nm should beinterpreted to include not only the explicitly recited limits of about 1nm and about 200 nm, but also to include individual sizes such as 2 nm,3 nm, 4 nm, and sub-ranges such as 10 nm to 50 nm, 20 nm to 100 nm, etc.. . .

The publications discussed or cited herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.All publications mentioned herein are incorporated herein by referenceto disclose and describe the structures and/or methods in connectionwith which the publications are cited. The following applications arealso incorporated herein by reference for all purposes: U.S. Pat. Nos.7,888,125, 8,007,999, 8,088,593 and U.S. Publication No., US20120309636,PCT Application No. PCT US2012/057155, U.S. patent application Ser. No.13/244,952, and PCT Application No. PCT/US2011/53188, filed Sep. 25,2011, U.S. patent application Ser. No. 13/244,946, filed Sep. 26, 2011,PCT Application No. PCT/US11/53189, filed Sep. 25, 2011, PatentCooperation Treaty Application No. PCT/US2011/53188; Patent CooperationTreaty Application No. PCT/US2012/57155; U.S. patent application Ser.No. 13/244,947; U.S. patent application Ser. No. 13/244,949; U.S. patentapplication Ser. No. 13/244,950; U.S. patent application Ser. No.13/244,951; U.S. patent application Ser. No. 13/244,952; U.S. patentapplication Ser. No. 13/244,953; U.S. patent application Ser. No.13/244,954; U.S. patent application Ser. No. 13/244,956; and U.S. patentapplication Ser. No. 13/769,820, entitled “Systems and Methods forMulti-Purpose Analysis,” filed Feb. 18, 2013, all of which applicationsare hereby incorporated by reference in their entireties. The followingapplications are fully incorporated herein by reference for allpurposes: U.S. Pat. No. 8,088,593; U.S. Pat. No. 8,380,541; U.S. patentapplication Ser. No. 13/769,798, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/769,779, filed Feb. 18, 2013; U.S. Pat. App.Ser. No. 61/766,113 filed Feb. 18, 2013, U.S. patent application Ser.No. 13/244,947 filed Sep. 26, 2011; PCT/US2012/57155, filed Sep. 25,2012; U.S. application Ser. No. 13/244,946, filed Sep. 26, 2011; U.S.patent application Ser. No. 13/244,949, filed Sep. 26, 2011; and U.S.Application Ser. No. 61/673,245, filed Sep. 26, 2011, U.S. PatentApplication Ser. No. 61/786,351 filed Mar. 15, 2013, U.S. PatentApplication Ser. No. 61/697,797 filed Sep. 6, 2012, and U.S. PatentApplication Ser. No. 61/733,886 filed Dec. 5, 2012, the disclosures ofwhich patents and patent applications are all hereby incorporated byreference in their entireties for all purposes.

While the above is a complete description of the preferred embodiment ofthe present invention, it is possible to use various alternatives,modifications and equivalents. Therefore, the scope of the presentinvention should be determined not with reference to the abovedescription but should, instead, be determined with reference to theappended claims, along with their full scope of equivalents. Anyfeature, whether preferred or not, may be combined with any otherfeature, whether preferred or not. The appended claims are not to beinterpreted as including means-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase“means for.” It should be understood that as used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. Also, as used in the description herein and throughout theclaims that follow, the meaning of “in” includes “in” and “on” unlessthe context clearly dictates otherwise. Finally, as used in thedescription herein and throughout the claims that follow, the meaningsof “and” and “or” include both the conjunctive and disjunctive and maybe used interchangeably unless the context expressly dictates otherwise.Thus, in contexts where the terms “and” or “or” are used, usage of suchconjunctions do not exclude an “and/or” meaning unless the contextexpressly dictates otherwise.

What is claimed is:
 1. A system for analyzing a sample, the systemcomprising: an illumination source; a sample holder configured toreceive light from the illumination source to provide both trans and epiillumination to the sample in the sample holder, wherein theillumination source is positioned to illuminate substantially only oneside of the sample holder.
 2. The system of any one of the foregoingclaims, wherein total internal reflection is used to provide transillumination.
 3. The system of any one of the foregoing claims, whereintotal internal reflection in the cuvette is used to provide transillumination.
 4. The system of any one of the foregoing claims, whereinthe sample holder comprises at least one or more opticallynon-transmissive surfaces.
 5. The system of any one of the foregoingclaims, wherein the sample holder comprises a cuvette.
 6. The system ofany one of the foregoing claims, wherein the cuvette comprises two ormore sample channels for holding sample.
 7. The system of any one of theforegoing claims, wherein the cuvette has a circular horizontal,cross-sectional shape.
 8. The system of any one of the foregoing claims,wherein the cuvette has a saw tooth vertical cross-sectional shape. 9.The system of any one of the foregoing claims, wherein the cuvette has astep-shaped vertical cross-sectional shape.
 10. The system of any one ofthe foregoing claims, wherein the sample holder comprises an opticallytransmissive surface configured to positioned at a first location to beilluminated by the illumination source.
 11. The system of any one of theforegoing claims further comprising an optical pathway from theillumination source to the sample holder wherein the pathway comprises aringlight.
 12. The system of any one of the foregoing claims furthercomprising an optical pathway from the illumination source to the sampleholder wherein the pathway comprises an LED based ringlight.
 13. Thesystem of any one of the foregoing claims further comprising an opticalpathway from the illumination source to the sample holder wherein thepathway comprises an laser based ringlight.
 14. The system of any one ofthe foregoing claims further comprising compression device for holdingthe sample holder against in a desired location for illumination by theillumination source.
 15. The system of any one of the foregoing claimsfurther comprising an optically clear surface shaped to engage anoptically clear surface of the sample holder.
 16. The system of any oneof the foregoing claims further comprising a detector configured toimage only a portion of a channel in the sample holder.
 17. The systemof any one of the foregoing claims further comprising a detectorconfigured to image an entire channel in the sample holder.
 18. Thesystem of any one of the foregoing claims wherein the sample holder isconfigured to hold the sample in a static, non-flowing manner duringimaging.
 19. The system of any one of the foregoing claims whereinduring imaging, the sample holder is configured to hold one portion ofthe sample in a static, non-flowing manner and another portion in aflowing manner.
 20. The system of any one of the foregoing claimswherein during imaging, the sample holder is configured to hold thesample in a flowing manner.
 21. The system of any one of the foregoingclaims wherein the sample remains separate from a detector in a fluidcircuit fully confined in the sample holder, where the sample holder ismovable relative to the detector.
 22. A method for the measurement of acomponent of interest in cells of a cellular population in a sample,comprising: a) obtaining a quantitative measurement of a marker presentin cells of the cellular population in the sample; b) based on themeasurement of part a), determining, with the aid of a computer, anapproximate amount of cells in the cellular population present in thesample; c) based on the results of part b), adding an amount of a cellmarker to add to the sample, wherein the marker binds specifically tothe component of interest in cells of the cellular population and isconfigured to be readily detectable; d) assaying cells in the sample formarker bound to the compound of interest; and e) based on the amount ofmarker bound to the compound of interest, determining the amount of thecomponent of interest in cells of the cellular population of the sample.23. A method for focusing a microscope, comprising: a) mixing a samplecontaining an object for microscopic analysis with a reference particlehaving a known size, to generate a mixture containing the sample andreference particle; b) positioning the mixture of step a) into a lightpath of a microscope; c) exposing the mixture of step a) to a light beamconfigured to visualize the reference particle; and d) focusing themicroscope based on the position of the reference particle within themixture.
 24. A method of identifying a cell in a sample containing aplurality of cells, comprising: (a) assaying a cell of the plurality ofcells for at least one of: (i) the presence of a cell surface antigen;(ii) the amount of a cell surface antigen; or (iii) cell size; (b)assaying the cell of (a) for at least one of: (i) nuclear size; or (ii)nuclear shape; and (c) assaying the cell of (a) and (b) for quantitativecell light scatter, wherein the combination of information from steps(a), (b), and (c) is used to identify the cell in the sample containinga plurality of cells